Design, synthesis and activity as acid ceramidase inhibitors of 2-oxooctanoyl and N-oleoylethanolamine analogues

Article abstract of DOI:10.1016/j.chemphyslip.2006.07.001

The synthesis of novel N-acylethanolamines and their use as inhibitors of the aCDase is reported here. The compounds are either 2-oxooctanamides or oleamides of sphingosine analogs featuring a 3-hydroxy-4,5-hexadecenyl tail replaced by ether or thioether

Full text of DOI:10.1016/j.chemphyslip.2006.07.001

Chemistry and Physics of Lipids 144 (2006) 69–84
Design, synthesis and activity as acid ceramidase inhibitors of
2-oxooctanoyl and N-oleoylethanolamine analogues
Santiago Grijalvoa,b, Carmen Bediaa, Gemma Triolaa, Josefina Casasa,
a
c
c
d
´
Amadeu Llebaria , Jordi Teixido , Obdulia Rabal , Thierry Levade ,
Antonio Delgadoa,b,∗, Gemma Fabrias
`
a,∗
a
Research Unit on Bioactive Molecules (RUBAM), Departament de Qu´ımica Orga`nica Biolo`gica, Institut d’Investigacions Qu´ımiques i
Ambientals de Barcelona (IIQAB-CSIC), Jordi Girona 18-26, E-08034 Barcelona, Spain
b
Universitat de Barcelona, Facultat de Farma`cia, Unitat de Qu´ımica Farmace`utica (Unitat Associada al CSIC),
Avda. Juan XXIII s/n, E-08028 Barcelona, Spain
Grup d’Enginyeria Molecular, Institut Qu´ımic de Sarria` (IQS), Universitat Ramon Llull, Via Augusta 390, E-08017 Barcelona, Spain
c
d
Laboratoire de Biochimie “Maladies Metaboliques”, INSERM U. 466, CHU Rangueil, 1 Avenue Jean Poulhes,
31059 Toulouse Cedex 9, France
Received 11 June 2006; accepted 12 July 2006
Available online 7 August 2006
Abstract
The synthesis of novel N-acylethanolamines and their use as inhibitors of the aCDase is reported here. The compounds are
either 2-oxooctanamides or oleamides of sphingosine analogs featuring a 3-hydroxy-4,5-hexadecenyl tail replaced by ether or
thioether moieties. It appears that, within the 2-oxooctanamide family, the C3–OH group of the sphingosine molecule is required for
inhibition both in vitro and in cultured cells. Furthermore, although the (E)-4 double bond is not essential for inhibitory activity, the
(E) configuration is required, since the analogue with a (Z)-4 unsaturation was not inhibitory. None of the oleamides inhibited the
aCDase in vitro. Conversely, with the exception of N-oleoylethanolamine and its analogs with S-decyl and S-hexadecyl substituents,
all the synthesized oleamides inhibited the aCDase in cultured cells, although with a relatively low potency. We conclude that
novel aCDase inhibitors can evolve from N-acylation of sphingoid bases with electron deficient-acyl groups. In contrast, chemical
modification of the N-oleoylsphingosine backbone does not seem to offer an appropriate strategy to obtain aCDase inhibitors.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Sphingolipids; Ceramide; Farber disease; Ceramidase; Inhibitor
1. Introduction
Abbreviations: aCDase, acid ceramidase; HRMS, high resolution-
mass spectrometry; IR, infrared; NOE, N-oleoylethanolamine; NMR,
affect the cell fate by producing a transient elevation
nuclear magnetic resonance; nCDase, neutral ceramidase
Stress stimuli and a number of extracellular agents
of the endogenous levels of ceramide. Exposure of
cells to radiation or chemotherapy is associated with
increased ceramide production due to the enhance-
ment of the de novo synthesis pathway, sphingomyelin
catabolism, or both. Ceramide can be metabolized into
less toxic forms by glycosylation, phosphorylation, or
∗
Corresponding authors at: Research Unit on Bioactive Molecules
(RUBAM), Departament de Qu´ımica Orga`nica Biolo`gica, Institut
d’Investigacions Qu´ımiques i Ambientals de Barcelona (IIQAB-
CSIC), Jordi Girona 18-26, E-08034 Barcelona, Spain.
Tel.: +34 93 4006115; fax: +34 93 2045904.
E-mail addresses: adcqob@iiqab.csic.es (A. Delgado),
gfdqob@iiqab.csic.es (G. Fabria`s).
0009-3084/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2006.07.001

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S. Grijalvo et al. / Chemistry and Physics of Lipids 144 (2006) 69–84
hydrolysis to sphingosine, which is in turn phospho-
rylated to the anti-apoptotic sphingosine-1-phosphate.
Several investigations have demonstrated that inhibition
of ceramide metabolization improve cancer chemother-
apy. For example, Reynolds et al. (2004) showed that
the combination of the synthetic retinoid fenretinide
with inhibitors of either sphingosine kinase or gluco-
sylceramide synthase increase antitumor activity in pre-
clinical models with minimal toxicity for non-malignant
cells. Moreover, blocking the conversion of ceramide
Fig. 1. Structures of NOE, 2-oxooctanamides and oleoylamides used in this work.

S. Grijalvo et al. / Chemistry and Physics of Lipids 144 (2006) 69–84
71
to glucosylceramide increases adriamicyn resistant cell
sensitivity to antitumoral agents (Lavie et al., 1997;
Lucci et al., 1999). In addition, inhibition of the acid
ceramidase (aCDase) prevents tumor growth and metas-
tasis of aggressive human colon cancer cell lines to the
liver (Selzner et al., 2001), sensitizes prostate tumors
to the effects of radiation (Samsel et al., 2004), and
induces apoptosis in human melanoma (Raisova et al.,
2002) and prostate cancer cells (Eto et al., 2006). Fur-
thermore, human aCDase is overexpressed in prostate
cancer and this enzyme may play an important role in
prostate carcinogenesis (Raisova et al., 2002). Strelow
et al. (2000) reported that overexpression of aCDase
protects from tumor necrosis factor-induced cell death.
This data supports a role of aCDase in signalling pro-
cesses by regulating the lysosomal pool of ceramide. It
also strengthens the notion that aCDase inhibitors are
potential antiproliferative and cytostatic drugs useful for
cancer chemotherapy.
Ceramidases hydrolyze the ceramide amide bond
yielding sphingosine and fatty acids. According to their
activity pH optimum, ceramidases are classified into
acid (aCDase), neutral (nCDase) (El Bawab et al., 1999,
2000; Tani et al., 2000a,b; Yoshimura et al., 2002, 2004)
and alkaline (Yada et al., 1995; Okino et al., 1998, 1999;
Mao et al., 2000a,b, 2001). The aCDase is localized
in the lysosomes. Acid ceramidase deficiencies cause
Farber’s disease or lipogranulomatosis (Bernardo et al.,
1995; Koch et al., 1996; Bar et al., 2001), a rare recessive
autosomal sphingolipidosis for which there is not known
treatment. In many sphingolipidosis variants, the mutant
enzymes retain catalytic activity, but are prone to fold
into improper conformations, which impair their trans-
port into the native cellular compartment for function.
The misfolded proteins are retained in the endoplasmic
reticulum and undergo premature degradation (Berg-
Fussman et al., 1993). The use of small molecules as
chemical chaperones that bind to the mutant protein and
templates its correct folding for further trafficking and
function is well documented (Morello et al., 2000). In
this context, competitive enzyme inhibitors have been
used as active-site-specific chaperones in lipid storage
disorders (Fan et al., 1999; Asano et al., 2000; Sawkar
et al., 2002; Lin et al., 2004), producing an increase
in the residual enzyme activity, which has a significant
impact on the disease outgrowth. In the light of these
evidences, aCDase inhibitors deserve attention not only
for their potential as chemotherapeutic drugs but also as
putative chemical chaperones for the treatment of Farber
disease.
tetradecanoylamino)-1-(4-nitrophenyl)-1,3-propanedi-
ol), which has exhibited interesting activities in can-
cer chemotherapy (Selzner et al., 2001; Raisova et
al., 2002; Samsel et al., 2004) and its N-tetradecyl
analog AD2646 (Granot et al., 2006). Furthermore,
N-oleoylethanolamine (NOE) has been extensively
used as aCDase inhibitor in basic studies (Castillo and
Teegarden, 2001; Friedrichs et al., 2002; Batra et al.,
2004), but its low potency and poor selectivity preclude
it from any therapeutic use. Finally, a 2-oxooctanamide
analog of the dihydroceramide desaturase inhibitor
GT11 (Triola et al., 2001) has been recently reported
(Bedia et al., 2005) as an efficient mechanism-based
inhibitor aCDase.
Based on the above precedents, the usefulness of
the oleamide and 2-oxooctanamide moieties as suit-
able frameworks for the synthesis of novel aCDase
inhibitors has been explored in this article. Specifically,
we report on the results obtained with a small com-
binatorial library of aminoethanols N-acylated with 2-
oxooctanoyl or oleoyl groups and branched at the C2
position of the aminoethanol core with a variety of R
substituents (Fig. 1).
2. Experimental procedures
2.1. General
Solvents were distilled prior to use and dried by stan-
dard methods (Perrin and Armarego, 1988). Unless oth-
erwise indicated, NMR spectra were recorded in CDCl3
at 300 MHz for 1H and 75 MHz for 13C. Chemical shifts
are reported in delta units (δ), parts per million (ppm)
relative to the singlet at 7.24 ppm of CDCl3 for 1H and
to the centre line of the triplet at 77.0 ppm of CDCl3 for
13C. IR spectra were measured in film. [α]D values are
given in 10−1 deg cm2 g−1
.
2.2. Synthesis of compounds
2.2.1.
4(S)-2,2-Dimethyl-4-(p-tolylsulfonyloxymethyl)
oxazolidine-3-carboxylic acid tert-butyl ester (26)
A
solution of 2,2-dimethyl-4-(hydroxymethyl)
oxazolidine-3-carboxylic acid tert-butyl ester (Garner
and Park, 1987) (2.24 g, 9.70 mmol) in CH2Cl2 was
added dropwise to a mixture of dimethylaminopyridine
(572 mg, 4.85 mmol) and TsCl (3.70 g, 19.4 mmol)
in CH2Cl2, followed by triethylamine (2.70 ml,
19.4 mmol) addition. After overnight stirring at room
temperature, the reaction mixture was washed with 1N
KHSO4 (3× 30 ml), and brine (3× 30 ml). The organic
The aCDase inhibitors reported to date are
scarce and include B13 compound ((1R,2R)-2-(N-

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S. Grijalvo et al. / Chemistry and Physics of Lipids 144 (2006) 69–84
layer was dried and the solvent was removed at reduced
pressure. The crude residue was purified by flash
chromatography (7:1 n-hexane/ethyl acetate) providing
tosylate 26 (3.35 g, 8.73 mmol, 90%) as a white solid:
mp 107–109 ◦C; lit (Porte et al., 1998) 108–109 ◦C;
33.9, 32.2, 31.0, 29.6, 29.4, 28.5, 28.3, 27.5, 26.9,
24.4, 23.0, 22.1, 14.0; the spectrum shows rotamers;
IR: 2950, 1740, 1352 cm−1; [α]D = −74.9 (c = 0.5,
CHCl3, 20 ◦C); HRMS—calculated for C16H31NO3S
[M + Na]+: 340.1923; found: 340.1916.
1
TLC Rf = 0.32; H NMR: δ 7.78 (d, J = 8.0 Hz, 2H),
7.25 (m, 2H), 4.20–3.60 (m, 5H), 2.41 (s, 3H), 1.45
(s, 3H), 1.42 (s, 3H), 1.39 (s, 9H); 13C NMR: δ 152.9,
142.0, 137.3, 130.5, 127.7, 126.9, 94.4, 81.6, 67.0,
64.5, 56.2, 28.4, 27.1, 23.6, 21.2; IR: 3030, 1710, 1400,
1370, 1355, 1150 cm−1; [α]D = −78.3 (c = 2.0, CHCl3,
20 ◦C); lit (Porte et al., 1998) −79.2 (c = 2.01, CHCl3,
25 ◦C).
2.3.3. 4(S)-4-Decylthiomethyl-2,2-
dimethyloxazolidine-3-carboxylic acid tert-butyl
ester (29)
Yield 82% after flash chromatography on hex-
anes (Rf = 0.27); 1H NMR: 3.90 (m, 3H), 2.90 (m,
2H), 2.45 (m, 2H), 1.57 (m, 3H), 1.42 (m, 14H),
1.22 (m, 14H), 0.84 (t, J = 6.6 Hz, 3H); 13C NMR:
150.4, 150.6, 93.0, 93.9, 79.9, 79.7, 66.1, 65.9, 57.9,
57.6, 34.4, 33.7, 32.2, 31.7, 29.8, 29.6, 29.4, 29.2,
29.0, 28.6, 28.4, 28.3, 27.5, 26.8, 24.2, 23.0, 22.4,
2.3. General procedure for the reaction of tosylate
26 with alcohols and thiols
14.0; IR: 2960, 1730, 1350 cm−1
;
[α]D = −61.8
A solution of the corresponding alcohol or thiol
(1.0 mmol) in dimethylformamide (1.0 ml) was added
dropwise to a suspension of NaH (42 mg of a 60% dis-
persioninmineraloil, equivalentto25 mg, 1.04 mmol)in
dimethylformamide (2.0 ml) at room temperature. After
ceasing gas evolution, the resulting mixture was trans-
ferred to a solution of tosylate 26 (200 mg, 0.52 mmol) in
dimethylformamide (3.0 ml). The reaction mixture was
stirred at 40 ◦C for 1 h, cooled to room temperature,
and treated with 1N HCl (5 ml). The organic phase was
extracted with diethyl ether (3× 20 ml), washed with
brine, dried and evaporated to dryness to yield an oil,
which was purified by flash chromatography to produce
compounds 27–37.
(c = 0.5,
CHCl3,
20 ◦C);
HRMS—calculated
for C21H41NO3S [M + Na]+: 410.2705; found:
410.2721.
2.3.4. 4(S)-4-Hexadecylthiomethyl-2,2-
dimethyloxazolidine-3-carboxylic acid tert-butyl
ester (30)
Yield 88% after flash chromatography on hexanes
(Rf = 0.3); 1H NMR: 3.87 (m, 3H), 2.84 (m, 2H),
2.45 (m, 3H), 1.54 (m, 4H), 1.46 (m, 12H), 1.23 (m,
26H), 0.84 (t, J = 6.6 Hz, 3H); 13C NMR: 152.0, 151.8,
95.1, 94.9, 80.1, 79.9, 66.4, 66.3, 57.3, 57.2, 34.4,
34.3, 32.1, 31.8, 29.9, 29.7, 29.6, 29.5, 29.4, 29.3,
29.1, 28.7, 28.4, 28.3, 27.5, 26.8, 24.2, 23.0, 22.6,
14.0; IR: 2954, 1738, 1355 cm−1
;
[α]D = −42.0
2.3.1. 4(S)-4-Ethylthiomethyl-2,2-
dimethyloxazolidine-3-carboxylic acid tert-butyl
ester (27)
(c = 1.0,
CHCl3,
20 ◦C);
HRMS—calculated
for C27H53NO3S [M + Na]+: 494.3644; found:
494.3631.
Yield 98% after flash chromatography on 30:1 hex-
1
anes/ethyl acetate (Rf = 0.27); H NMR: 3.86 (m, 3H),
2.3.5. 4(S)-4-(4-Chlorobenzylthiomethyl)-2,2-
dimethyloxazolidine-3-carboxylic acid tert-butyl
ester (31)
2.82 (m, 2H), 2.50 (m, 3H), 1.52 (m, 3H), 1.42
(m, 14H), 1.22 (t, J = 6.6 Hz, 3H); 13C NMR: 152.0,
150.5, 94.1, 93.9, 80.1, 80.0, 66.4, 66.2, 56.5, 56.3,
34.0, 32.7, 28.3, 27.9, 27.6, 26.0, 24.1, 22.5, 14.5;
IR: 2950, 1738, 1354 cm−1; [α]D = −40.4 (c = 4.5,
CHCl3, 20 ◦C); HRMS—calculated for C13H25NO3S
[M + Na]+: 298.1453; found: 298.1457.
Yield 98% after flash chromatography on 25:1 hex-
anes/ethyl acetate (Rf = 0.34); mp 74 ◦C; 1H NMR: 7.25
(m, 5H), 4.20–3.80 (m, 3H), 3.69 (s, 2H), 2.79 (m, 1H),
2.49 (m, 1H), 1.58 (s, 3H), 1.52 (s, 3H), 1.48 (s, 9H), 1.44
(s, 3H), 1.40 (s, 3H); the spectrum shows rotamers; 13
C
NMR: 151.9, 151. 7, 137.0, 136.6, 132.7, 132.6, 130.7,
129.9, 128.5, 128.4, 94.1, 93.6, 80.2, 79.8, 66.4, 56.9,
56.7, 36.0, 35.3, 34.6, 33.1, 28.3, 27.5, 26.7, 24.2, 23.0;
IR: 2954, 1717, 1656, 1592 1355 cm−1; [α]D = −128.9
(c = 1.0, CHCl3, 20 ◦C); HRMS—calculated for
2.3.2. 4(S)-2,2-Dimethyl-4-(pentylthiomethyl)
oxazolidine-3-carboxylic acid tert-butyl ester (28)
Yield 99% after flash chromatography on 45:1 hex-
1
anes/ethyl acetate (Rf = 0.27); H NMR: 3.92 (m, 3H),
2.82 (m, 2H), 2.50 (m, 2H), 1.54 (m, 3H), 1.44 (m,
18H), 0.84 (t, J = 6.6, 3H); 13C NMR: 152.1, 151.9,
94.0, 93.9, 80.2, 80.0, 66.1, 66.0, 57.5, 57.3, 34.5,
C18H26ClNO3S
394.1235.
[M + Na]+:
394.1220;
found:

S. Grijalvo et al. / Chemistry and Physics of Lipids 144 (2006) 69–84
73
2.3.6. 4(S)-2,2-Dimethyl-4-(2-
naphthylthiomethyl)oxazolidine-3-carboxylic acid
tert-butyl ester (32)
2.3.9. 4(R)-4-((4-Chloro-3-methylphenoxy)methyl)-
2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl
ester (35)
Yield 95% after flash chromatography on 30:1 hex-
anes/ethyl acetate (Rf = 0.35); mp 75 ◦C; 1H NMR: 8.04
(s, 1H), 7.90–7.76 (m, 3H), 7.46 (m, 3H), 4.20–3.90
(m, 3H), 3.66 (m, 1H), 3.42 (m, 1H), 2.90 (m, 2H),
1.64 (s, 3H), 1.62 (s, 3H), 1.52 (s, 9H), 1.48 (s, 3H),
1.46 (s, 3H), 1.38 (s, 9H); 13C NMR: 152.0, 151.4,
134.0, 133.6, 133.0, 132.2, 132.0, 131.4, 128.8, 128.6,
128.4, 127.8, 127.6, 127.5, 127.3, 127.2, 126.6, 126.4,
126.2, 126.0, 125.3, 125.1, 94.3, 93.8, 80.5, 80.1, 66.0,
56.5, 35.9, 33.4, 28.4, 27.8, 26.9, 24.2, 23.0; IR: 2954,
1738, 1717, 1592 1535 cm−1; [α]D = +126.5 (c = 1.0,
CHCl3, 20 ◦C); HRMS—calculated for C21H27NO3S
[M + Na]+: 396.1610; found: 396.1621.
Yield 76% after flash chromatography on 30:1
hexanes/ethyl acetate (Rf = 0.23); 1H NMR: 7.21 (d,
J = 8.8 Hz, 2H), 6.79 (m, 2H), 6.72 (m, 2H), 4.25
(m, 1H), 4.15 (m, 2H), 4.05 (m, 3H), 3.99 (m,
2H), 3.81 (m, 2H), 2.32 (s, 6H), 1.62 (m, 6H),
1.49 (m, 24H); the spectrum shows rotamers; 13C
NMR: 156.9, 156.8, 152.3, 152.2, 137.0, 136.8, 129.4,
125.8, 117.1, 113.0, 112.8, 94.0, 93.4, 80.5, 80.1,
66.8, 66.2, 65.2, 65.0, 55.9, 55.7, 29.6, 28.4, 28.3,
27.4, 26.7, 24.1, 22.9, 20.1; the spectrum shows
rotamers; IR: 2965, 2366, 1694, 1596, 1479, 1426, 1386,
1240, 1171, 1084, 1039 cm−1; [α]D = −47.4 (c = 1,
CHCl3, 20 ◦C); HRMS—calculated for C18H26ClNO4
[M + Na]+: 378.1448; found: 378.1457.
2.3.7. 4(R)-4-((4-(2-Methoxyethyl)phenoxy)
methyl)-2,2-dimethyloxazolidine-3-carboxylic acid
tert-butyl ester (33)
2.3.10. 4(R)-2,2-Dimethyl-4-((4-propylphenoxy)
methyl)oxazolidine-3-carboxylic acid tert-butyl
ester (36)
Yield 71% after flash chromatography on 30:1 hex-
Yield 83% after flash chromatography on 40:1
hexanes/ethyl acetate (Rf = 0.23); 1H NMR: 7.07 (d,
J = 8.2 Hz, 4H), 6.86 (m, 4H), 4.28 (m, 1H), 4.18 (m,
2H), 4.10 (m, 4H), 3.99 (m, 3H), 3.82 (t, J = 7.7 Hz, 4H),
2.52 (m, 4H), 1.62 (s, 3H), 1.56 (s, 3H), 1.49 (s, 30H),
0.92 (t, J = 7.7 Hz, 6H); the spectrum shows rotamers;
13C NMR: 156.5, 156.3, 152.1, 151.8, 135.1, 134.9,
129.2, 114.3, 114.2, 94.0, 93.4, 80.3, 80.0, 66.6, 66.0,
65.3, 65.1, 56.0, 55.7, 37.0, 29.6, 28.4, 28.3, 27.4, 26.7,
24.6, 24.2, 22.9, 13.6; the spectrum shows rotamers; IR:
1
anes/ethyl acetate (Rf = 0.23); H NMR: 7.13 (m, 4H),
6.87 (m, 4H), 4.26 (m, 1H), 4.18 (m, 2H), 4.08 (m, 3H),
3.98 (m, 2H), 3.81 (m, 2H), 3.55 (t, J = 6.6 Hz, 4H), 3.34
(s, 6H), 2.81 (t, J = 6.6 Hz, 4H), 1.61 (s, 3H), 1.56 (s,
3H), 1.48 (s, 30H); the spectrum shows rotamers; 13C
NMR: 156.9, 156.8, 152.1, 151.6, 131.4, 131.0, 129.6,
114.5, 114.4, 93.9, 93.4, 80.3, 80.1, 73.7, 66.5, 66.0,
65.3, 65.1, 58.5, 55.9, 55.6, 35.1, 29.6, 28.4, 28.3, 27.4,
26.7, 24.2, 22.9; IR: 2978, 2934, 1698, 1508, 1388,
1244, 1171, 1113 cm−1; [α]D = −40.4 (c = 1, CHCl3,
20 ◦C); HRMS—calculated for C20H31NO5 [M + Na]+:
388.2100; found: 388.2090.
2977, 2370, 1703, 1507, 1365, 1232, 1167, 1082 cm−1
;
[α]D = −37.3 (c = 1, CHCl3, 20 ◦C); HRMS—calculated
for C20H31NO4 [M + Na]+: 372.2151; found:
372.2144.
2.3.8. 4(R)-4-((4-Benzylphenoxy)methyl)-2,2-
dimethyloxazolidine-3-carboxylic acid tert-butyl
ester (34)
2.3.11. 4(R)-4-((4-(Decyloxy)phenoxy)methyl)-2,2-
dimethyloxazolidine-3-carboxylic acid tert-butyl
ester (37)
Yield 84% after flash chromatography on 25:1 hex-
1
anes/ethyl acetate (Rf = 0.23); H NMR: 7.27 (m, 2H),
Yield 75% after flash chromatography on 30:1 hex-
anes/ethyl acetate (Rf = 0.33); 1H NMR (500 MHz): 6.88
(m, 4H), 6.83 (m, 4H), 4.28 (m, 2H), 4.17 (m, 2H),
4.12 (m, 2H), 4.07 (m, 2H), 4.00 (m, 2H), 3.91 (m, 4H),
3.80 (m, 2H), 1.76 (m, 4H), 1.63 (s, 3H), 1.51 (s, 12H),
1.28 (m, 28H), 0.89 (t, J = 6.3 Hz, 6H); the spectrum
shows rotamers; 13C NMR (125 MHz): 153.5, 153.3,
152.5, 152.4, 152.2, 151.6, 115.4, 115.3, 93.9, 93.4,
80.3, 80.0, 68.5, 67.3, 66.6, 65.3, 65.1, 56.0, 55.8, 31.8,
29.6, 29.5, 29.4, 29.3, 29.2, 28.4, 28.3, 27.4, 26.7, 25.9,
24.2, 23.0, 22.6, 14.0; the spectrum shows rotamers; IR:
2926, 2852, 2370, 2341, 1704, 1510, 1468, 1389, 1365,
1232, 1082, 825 cm−1; [α]D = −36.8 (c = 1, CHCl3,
7.18 (m, 2H), 7.09 (m, 2H), 6.87 (m, 2H), 4.28 (m, 1H),
4.18 (m, 2H), 4.09 (m, 4H), 3.99 (m, 2H), 1.62 (s, 3H),
1.56 (s, 3H), 1.49 (s, 15H), 1.48 (s, 15H); the spectrum
shows rotamers; 13C NMR: 156.7, 156.6, 152.2, 151.6,
141.5, 141.4, 133.6, 133.3, 129.8, 128.7, 128.3, 125.8,
114.5, 93.9, 93.4, 80.3, 80.1, 66.6, 66.0, 65.3, 65.1, 55.9,
55.7, 40.9, 29.6, 28.4, 28.3, 27.4, 26.7, 24.2, 23.0; the
spectrum shows rotamers; IR: 2977, 2368, 2328, 1702,
1612, 1507, 1388, 1365, 1232, 1167, 1082, 1036 cm−1
;
[α]D = −50.1 (c = 1, CHCl3, 20 ◦C); HRMS—calculated
for C24H31NO4 [M + Na]+: 420.2151; found:
420.2159.

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S. Grijalvo et al. / Chemistry and Physics of Lipids 144 (2006) 69–84
20 ◦C); HRMS—calculated for C27H45NO5 [M + Na]+:
486.3196; found: 486.3186.
2.4.3. (1ꢀS,2ꢀ)-N-(2-Hydroxy-1-hydroxymethyl-3-
heptadecynyl)-2-oxooctanamide
(14)
Yield 74%; 1H NMR: 7.66 (1H, J = 8.4 Hz, NH);
4.63 (m, 1H, H3), 4.13 (dd, 1H, J = 11.4 Hz, Jꢀ = 3.9 Hz,
H1), 4.0 (m, 1H, H2), 3.77 (dd, 1H, J = 11.1 Hz,
Jꢀ = 4.2 Hz, H1), 2.93 (t, 2H, J = 8.1 Hz), 2.67 (bs, 1H,
OH), 2.21 (dt, 2H, J = 6.9 Hz, Jꢀ = 2.1 Hz,), 1.60 (t, 2H,
J = 7.2 Hz), 1.50 (t, 2H, J = 7.2 Hz), 1.2–1.4 (m, 30
H), 0.87 (t, 6H, J = 6.9 Hz); 13C NMR: 198.6. 160.5,
122.3, 88.8, 62.2, 64.0, 54.8, 36.8, 31.9, 28.9, 31.5,
28.7, 29.6, 29.4, 28.4, 29.3, 29.1, 22.6, 22.4, 18.6,
23.0, 13.9, 14.1; IR: 3312, 2923, 1719, 1665, 1536,
1463, 1384, 1064 cm−1; [α]D = −1.3 (c = 0.63, CHCl3,
20 ◦C); HRMS—calculated for C26H47NO4 [M + Na]+:
460.3403; found: 460.3417.
2.4. General method for the synthesis of
α-ketoamides 12–15
To a solution of the corresponding sphingoid
aminoalcohol (0.1 mmol), 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (0.15 mmol) and 1-
hydroxybenzotriazole hydrate (0.15 mmol) in tetrahy-
drofurane was added a solution of the required car-
boxylic acid (0.15 mmol) in CH2Cl2 (2 ml). The reaction
mixture was stirred at room temperature for 12 h, diluted
with CH2Cl2 (10 ml), washed with brine and dried. Sol-
vent was removed under vacuum and purification of the
residue by flash chromatography eluting with a mixture
of CH2Cl2–MeOH (97:3) furnished pure compounds
12–15 as oils.
2.4.4. (1ꢀS,2ꢀR)-N-(2-Hydroxy-1-
hydroxymethylheptadecyl)-2-oxooctanamide (15)
Yield 69%; 1H NMR: 7.72 (1H, J = 7.2 Hz, NH);
4.0 (d, 1H, J = 9.3 Hz), 3.79 (m, 3H), 2.93 (t, 2H,
J = 7.5 Hz), 2.61 (bs, 1H, OH), 2.53 (bs, 1H, OH), 1.58
(m, 2H), 1.2–1.4 (m, 30 H), 0.87 (t, 6H, J = 6.3 Hz);
13C NMR: 198.6, 160.5, 29.6, 29.5, 73.7, 61.9, 53.8,
36.8, 31.9, 34.4, 31.5, 28.7, 25.8, 29.4, 29.3, 22.6,
22.4, 23.1, 14.1, 14.0; IR: 3290, 2934, 1658, 1532,
1462, 1068 cm−1; [α]D = +4.54 (c = 0.35, CHCl3,
20 ◦C); HRMS—calculated for C26H51NO4 [M + Na]+:
464.3716; found: 464.3725.
2.4.1. (E)-(1ꢀS,2ꢀR)-N-(2-Hydroxy-1-
hydroxymethyl-3-heptadecenyl)-2-oxooctanamide
(12)
Yield 71%; 1H NMR: 7.69 (1H, J = 8.7 Hz, NH);
5.78 (dt, 1H, J = 15.3 Hz, Jꢀ = 6.9 Hz), 5.52 (dd, 1H,
J = 15.1 Hz, Jꢀ = 5.1 Hz), 4.32 (t, 1H, 5.1 Hz, H3), 3.98
(dd, 1H, J = 11.4 Hz, Jꢀ = 3.9 Hz, H1), 3.86 (m, 1H, H2),
3.71 (dd, 1H, J = 11.4, Jꢀ = 3.3 Hz, H1), 2.90 (t, 2H,
J = 7.2 Hz), 2.03 (dd, 2H, J = 13.2, Jꢀ = 6.3 Hz), 1.59
(m, 4H), 1.2–1.4 (m, 30 H), 0.87 (t, 6H, J = 6.9 Hz);
13C NMR: 198.8, 160.3, 134.8, 128.3, 74.0, 61.8, 54.4,
36.8, 32.2, 31.9, 31.5, 29.6, 29.5, 29.4, 29.3, 29.1, 29.0,
22.6, 28.7, 23.1, 22.4, 14.1, 13.9; IR: 3264, 2923, 1662,
1461, 1373, 1036 cm−1; [α]D = +7.53 (c = 0.62, CHCl3,
20 ◦C); HRMS—calculated for C26H49NO4 [M + Na]+:
462.3560; found: 462.3550.
2.5. General method for the synthesis of 1–11 and
16–25 by deprotection-acylation of 27–37
A solution of the starting oxazolidines 27–37
(1.0 mmol) in CH2Cl2 (2.0 ml) was treated with triflu-
oroacetic acid (1.0 ml of a 20% solution in CH2Cl2).
The reaction mixture was stirred for 5 min at room
temperature and the solvent removed under vacuum
to afford crude aminoalcohols, as their trifluoroacetate
salts, which were submitted to acylation without fur-
ther purification. A solution of the crude trifluoroac-
etate in CH2Cl2 (2.0 ml) was treated with triethylamine
(2.5 mmol), followed by dropwise addition of a solu-
tion of the corresponding carboxylic acid (1.8 mmol),
1-hydroxybenzotriazole hydrate (1.8 mmol) and 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride (1.8 mmol) in CH2Cl2 (2.0 ml). After stirring for
16 h at room temperature, the organic phase was washed
with 3× 10 ml of 1N HCl solution and brine (3× 10 ml).
After the usual work-up, the crude residue was taken
up in CH2Cl2 (5.0 ml), treated with Amberlite A-21
(20 equiv., L = 4.7 mequiv./g), and stirred at room tem-
2.4.2. (Z)-(1ꢀS,2ꢀR)-N-(2-Hydroxy-1-
hydroxymethyl-3-heptadecenyl)-2-oxooctanamide
(13)
1
Yield 78%; H NMR: 0.87 (t, 6H, J = 6.9 Hz), 7.62
(1H, J = 8.1 Hz, NH); 5.59 (m, 1H), 5.48 (dd, 1H,
J = 9.9 Hz, Jꢀ = 8.4 Hz), 4.69 (m, 1H, H3), 4.04 (dd,
1H, J = 11.1 Hz, Jꢀ = 3.3 Hz, H1), 3.81 (m, 1H, H2),
3.74 (dd, 1H, J = 11.4 Hz, Jꢀ = 3.3 Hz, H1), 2.91 (t, 2H,
J = 7.2 Hz), 2.65 (bs, 1H, OH), 2.42 (bs, 1H, OH), 2.1
(m, 2H), 1.61 (m, 4H), 1.2–1.4 (m, 30 H); 13C NMR:
198.8, 160.3, 135.1, 128.1, 69.2, 62.0, 54.7, 36.8, 31.9,
31.5, 29.6, 26.8, 29.5, 29.4, 29.3, 29.2, 28.7, 28.2,
27.9, 27.8, 23.1, 22.6, 22.4, 17.5, 14.1, 13.9; IR: 3365,
2933, 1658, 1462, 1022.7 cm−1; [α]D = −5.21 (c = 0.69,
CHCl3, 20 ◦C); HRMS—calculated for C26H49NO4
[M + Na]+: 462.3560; found: 462.3546.

S. Grijalvo et al. / Chemistry and Physics of Lipids 144 (2006) 69–84
75
2.5.4. (2ꢀS)-N-(1-(Hexadecylthio)-3-
perature for 3 h. Filtration and evaporation produced an
oily residue, which was purified by flash chromatogra-
phy.
hydroxypropan-2-yl)-2-oxooctanamide (4)
Yield 42% after flash chromatography on 3:1 hex-
1
anes/ethyl acetate (Rf = 0.35); H NMR: 7.41 (broad,
1H), 4.03 (m, 1H), 3.87 (dd, J = 11.1 Hz, Jꢀ = 4.6 Hz,
1H), 3.75 (dd, J = 11.1 Hz, Jꢀ = 4.0 Hz, 1H), 2.91
(t, J = 7.1 Hz, 2H), 2.78 (dd, J = 13.5 Hz, Jꢀ = 6.7 Hz,
1H), 2.71 (dd, J = 13.5 Hz, Jꢀ = 6.4 Hz, 1H), 2.54 (t,
J = 7.1 Hz, 2H), 2.17 (broad, 1H), 1.60 (m, 4H),
1.25 (m, 32H), 0.87 (m, 6H); 13C NMR: 198.7,
160.1, 63.2, 50.7, 36.6, 32.7, 32.6, 31.8, 31.4, 29.5,
29.5, 29.5, 29.4, 29.4, 29.2, 29.1, 28.7, 28.6, 23.0,
22.5, 22.3, 14.0, 13.9; IR: 3315, 2925, 2853, 2375,
1717, 1656, 1521, 1466, 1033 cm−1; [α]D = +9.0 (c = 1,
CHCl3, 20 ◦C); HRMS—calculated for C27H53NO3S
[M + Na]+: 494.3644; found: 494.3649.
2.5.1. (2ꢀS)-N-(1-(Ethylthio)-3-hydroxypropan-2-
yl)-2-oxooctanamide (1)
Yield 67% after flash chromatography on 3:1
hexanes/acetone (Rf = 0.35); 1H NMR: 7.42 (broad,
1H), 4.04 (m, 1H), 3.88 (dd, J = 11.1 Hz, Jꢀ = 4.5 Hz,
1H), 3.76 (dd, J = 11.1 Hz, Jꢀ = 4.0 Hz, 1H), 2.91 (t,
J = 7.2 Hz, 2H), 2.77 (m, 2H), 2.58 (q, J = 7.4 Hz,
2H), 1.62 (m, 2H), 1.29 (m, 8H), 0.88 (m, 6H);
13C NMR: 199.0, 160.4, 63.4, 51.0, 36.9, 32.5,
31.7, 28.9, 26.7, 23.3, 22.6, 14.9, 14.2; IR: 3320,
2011, 2852, 2386, 1725, 1658, 1534, 1465, 1040 cm−1
;
[α]D = +5.9 (c = 1.0, CHCl3, 20 ◦C); HRMS—calculated
for C13H25NO3S [M + Na]+: 298.1453; found:
298.1447.
2.5.5. (2ꢀS)-N-(1-(4-Chlorobenzylthio)-3-
hydroxypropan-2-yl)-2-oxooctanamide (5)
Yield 69% after flash chromatography on 3:1 hex-
2.5.2. (2ꢀS)-N-(1-Hydroxy-3-(pentylthio)propan-2-
yl)-2-oxooctanamide (2)
1
anes/acetone (Rf = 0.33); H NMR: 4.02 (m, 1H), 3.82
(dd, J = 11.1 Hz, Jꢀ = 4.4 Hz, 1H), 3.70 (dd, J = 11.1 Hz,
Jꢀ = 4.0 Hz, 1H), 3.70 (s, 2H), 2.90 (t, J = 7.6 Hz, 2H),
2.62 (m, 2H), 2.17 (broad, 1H), 1.60 (m, 2H), 1.29 (m,
8H), 0.88 (t, J = 6.7 Hz, 3H); 13C NMR: 199.0, 160.4,
136.5, 130.5, 128.9, 63.2, 50.5, 37.0, 35.9, 32.0, 31.7,
28.9, 23.3, 22.6, 14.2; IR: 3344, 2923, 2861, 2388,
1721, 1673, 1521, 1093, 1042 cm−1; [α]D = +9.0 (c = 1,
CHCl3, 20 ◦C); HRMS—calculated for C18H26ClNO3S
[M + Na]+: 394.1220; found: 394.1232.
Yield 60% after flash chromatography on 4:1
hexanes/acetone (Rf = 0.33); 1H NMR: 7.42 (broad,
1H), 4.03 (m, 1H), 3.86 (dd, J = 11.3 Hz, Jꢀ = 4.4 Hz,
1H), 3.74 (dd, J = 11.3 Hz, Jꢀ = 3.7 Hz, 1H), 2.90 (t,
J = 7.1 Hz, 2H), 2.77 (dd, J = 13.6 Hz, Jꢀ = 6.9 Hz,
1H), 2.73 (dd, J = 13.6 Hz, Jꢀ = 6.5 Hz, 1H), 1.58
(m, 4H), 1.30 (m, 12H), 0.89 (m, 6H); 13C NMR:
199.0, 160.4, 63.4, 51.0, 36.9, 33.0, 32.9, 31.7,
31.1, 29.4, 28.9, 23.3, 22.6, 22.4, 14.2, 14.1; IR:
3120, 2856, 1710, 1663. 1535, 1475, 1035 cm−1
;
2.5.6. (2ꢀS)-N-(1-Hydroxy-3-(2-naphylthio)propan-
2-yl)-2-oxooctanamide (6)
[α]D = +2.2 (c = 1.0, CHCl3, 20 ◦C); HRMS—calculated
for C16H31NO3S [M + Na]+: 340.1922; found:
340.1912.
Yield 61% after flash chromatography on 3:1 hex-
1
anes/acetone (Rf = 0.35); H NMR: 7.89 (s, 1H), 7.78
(m, 3H), 7.46 (m, 3H), 4.11 (m, 1H), 3.93 (m,
1H), 3.75 (m, 1H), 3.31 (dd, J = 13.9 Hz, Jꢀ = 6.2 Hz,
1H), 3.24 (dd, J = 13.9 Hz, Jꢀ = 7.1 Hz, 1H), 2.81 (t,
J = 6.3 Hz, 2H), 1.65 (broad, s, 1H), 1.51 (m, 2H),
1.27 (m, 8H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR:
198.8, 160.3, 133.9, 132.5, 132.1, 129.0, 128.0, 127.9,
127.5, 127.4, 126.9, 126.2, 62.9, 51.2, 36.9, 34.2,
31.7, 31.1, 28.9, 23.2, 22.6, 14.2; IR: 3341, 2928,
2387, 1718, 1675, 1043, 810 cm−1; [α]D = +30.3 (c = 1,
CHCl3, 20 ◦C); HRMS—calculated for C21H27NO3S
[M + Na]+: 396.1610; found: 396.1512.
2.5.3. (2ꢀS)-N-(1-(Decylthio)-3-hydroxypropan-2-
yl)-2-oxooctanamide (3)
Yield 63% after flash chromatography on 4:1
hexanes/acetone (Rf = 0.38); 1H NMR: 7.41 (broad,
1H), 4.05 (m, 1H), 3.89 (m, 1H), 3.79 (m, 1H), 2.93
(t, J = 7.3 Hz, 2H), 2.79 (dd, J = 13.6 Hz, Jꢀ = 5.6 Hz,
1H), 2.74 (dd, J = 13.6 Hz, Jꢀ = 6.4 Hz, 1H), 2.56 (t,
J = 7.4 Hz, 2H), 2.24 (broad, 1H), 1.58 (m, 8H), 1.32 (m,
16H), 0.89 (t, J = 7.1 Hz, 6H); 13C NMR: 198.7, 160.1,
63.2, 50.7, 36.6, 32.7, 32.6, 31.8, 31.4, 29.6, 29.5, 29.4,
29.2, 29.1, 28.7, 28.6, 23.0, 22.5, 22.3, 14.0, 13.9; IR:
3324, 2915, 2853, 1715, 1654, 1534, 1470, 1044 cm−1
;
2.5.7. (2ꢀS)-N-(1-Hydroxy-3-(4-(2-methoxyethyl)
phenoxy)propan-2-yl)-2-oxooctanamide (7)
[α]D = +8.5 (c = 1.0, CHCl3, 20 ◦C); HRMS—calculated
for C21H41NO3S [M + Na]+: 410.2705; found:
410.2715.
Yield 58% after flash chromatography on 3:1 hex-
1
anes/acetone (Rf = 0.35); H NMR: 7.55 (broad, 1H),

76
S. Grijalvo et al. / Chemistry and Physics of Lipids 144 (2006) 69–84
7.53 (broad, 1H), 7.15 (d, J = 8.7 Hz, 2H), 6.84 (d,
J = 8.7 Hz, 2H), 4.26 (m, 1H), 4.16 (dd, J = 9.5 Hz,
Jꢀ = 4.2 Hz, 1H), 4.08 (dd, J = 9.6 Hz, Jꢀ = 5.0 Hz, 1H),
3.95 (dd, J = 11.2 Hz, Jꢀ = 4.5 Hz, 1H), 3.84 (dd,
J = 11.2 Hz, Jꢀ = 4.5 Hz, 1H), 3.56 (t, J = 7.1 Hz, 2H),
3.34 (s, 3H), 2.92 (t, J = 7.2 Hz, 2H), 2.82 (t, J = 7.0 Hz,
2H), 1.61 (m, 2H), 1.30 (m, 6H), 0.88 (t, J = 7.2 Hz, 3H);
13CNMR:198.9, 160.5, 156.7, 132.2, 130.1, 114.6, 73.9,
67.0, 62.4, 58.9, 50.7, 37.0, 35.5, 31.7, 28.9, 23.3, 22.6,
14.2; IR: 3335, 2928, 2862, 2367, 1712, 1688, 1513,
1469, 1240, 1115 cm−1; [α]D = −2.8 (c = 1, CHCl3,
20 ◦C); HRMS—calculated for C20H31NO5 [M + Na]+:
388.2100; found: 388.2107.
(500 MHz): 7.54 (broad, 1H), 6.83 (m, 4H), 4.24
(m, 1H), 4.13 (dd, J = 9.5 Hz, Jꢀ = 4.2 Hz, 1H), 4.04
(dd, J = 9.6 Hz, Jꢀ = 4.9 Hz, 1H), 3.95 (dd, J = 11.4 Hz,
Jꢀ = 4.5 Hz, 1H), 3.89 (t, J = 6.6 Hz, 2H), 3.83 (dd,
J = 11.2 Hz, Jꢀ = 5.0 Hz, 1H), 2.92 (t, J = 7.5 Hz, 2H),
1.74 (m, 2H), 1.60 (m, 2H), 1.43 (m, 2H), 1.29 (m,
18H), 0.87 (t, J = 7.1 Hz, 6H); 13C NMR (125 MHz):
198.9, 160.5, 154.1, 152.2, 115.7, 115.6, 68.8, 67.8, 62.5,
50.7, 37.0, 32.1, 31.7, 29.8, 29.7, 29.6, 29.5, 28.9, 26.2,
23.3, 22.9, 22.6, 14.3, 14.2; IR: 3271, 2920, 2847, 1707,
1659, 1507, 1242, 1049, 829 cm−1; [α]D = −10.2 (c = 1,
CHCl3, 20 ◦C); HRMS—calculated for C27H45NO5
[M + Na]+: 486.3195; found: 486.3204.
2.5.8. (2ꢀS)-N-(1-(4-Chloro-3-methylphenoxy)-3-
hydroxypropan-2-yl)-2-oxooctanamide (8)
2.5.11. (2ꢀS)-N-(1-(4-Benzylphenoxy)-3-
hydroxypropan-2-yl)-2-oxooctanamide (11)
Yield 59% after flash chromatography on 3:1 hex-
anes/acetone (Rf = 0.32); H NMR: 7.51 (broad, 1H),
Yield 59% after flash chromatography on 3:1 hex-
anes/acetone; mp 81–82 ◦C; (Rf = 0.31); 1H NMR: 7.53
(broad, 1H), 7.30 (m, 2H), 7.21 (m, 1H), 7.18 (m, 1H),
7.12 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 4.27
(m, 1H), 4.17 (dd, J = 9.6 Hz, Jꢀ = 4.2 Hz, 1H), 4.09 (dd,
J = 9.5 Hz, Jꢀ = 5.0 Hz, 1H), 3.96 (m, 1H), 3.93 (s, 2H),
3.84 (m, 1H), 2.92 (t, J = 7.3 Hz, 2H), 2.28 (broad, 1H),
1
7.23 (d, J = 8.5 Hz, 1H), 6.79 (d, J = 2.7 Hz, 1H), 6.69
(dd, J = 8.7 Hz, Jꢀ = 3.5 Hz, 1H), 4.26 (m, 1H), 4.15
(dd, J = 9.5 Hz, Jꢀ = 4.3 Hz, 1H), 4.06 (dd, J = 9.4 Hz,
Jꢀ = 5.1 Hz, 1H), 3.96 (m, 1H), 3.84 (m, 1H), 2.92 (t,
J = 7.2 Hz, 2H), 2.33 (s, 3H), 2.23 (broad, 1H), 1.59 (m,
2H), 1.30 (m, 6H), 0.88 (t, J = 7.2 Hz, 3H); 13C NMR:
198.9, 160.5, 156.8, 137.5, 130.0, 129.9, 126.9, 117.3,
113.2, 66.9, 62.1, 50.6, 37.0, 31.7, 28.9, 23.3, 22.6, 20.5,
14.2; IR: 3329, 2957, 2919, 2856, 2349, 1747, 1661,
1573, 1482, 1295, 1177, 1067 cm−1; [α]D = +5.2 (c = 1,
CHCl3, 20 ◦C); HRMS—calculated for C18H26ClNO4
[M + Na]+: 378.1449; found: 378.1456.
1.59 (m, 2H), 1.31 (m, 6H), 0.89 (t, J = 6.9 Hz, 3H); 13
C
NMR: 198.9, 160.5, 141.5, 134.4, 130.2, 129.0, 128.6,
126.2, 114.7, 67.0, 62.4, 50.6, 41.2, 37.0, 31.7, 28.9,
23.3, 22.6, 14.2; IR: 3322, 2923, 2359, 2340, 1721, 1652,
1506, 1244, 1079 cm−1; [α]D = −2.8 (c = 1, CHCl3,
20 ◦C); HRMS—calculated for C24H31NO4 [M + Na]+:
420.2151; found: 420.2164.
2.5.9. (2ꢀS)-N-(1-Hydroxy-3-(4-
2.5.12. (2ꢀS,9E)-N-(1-(Ethylthio)-3-hydroxypropan-
2-yl)octadec-9-enamide (16)
propylphenoxy)propan-2-yl)-2-oxooctanamide (9)
Yield 67% after flash chromatography on 3:1 hex-
anes/acetone; mp 55–56 ◦C; (Rf = 0.34); 1H NMR: 7.54
(broad, 1H), 7.11 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.6 Hz,
2H), 4.27 (m, 1H), 4.18 (dd, J = 9.5 Hz, Jꢀ = 4.2 Hz, 1H),
4.10 (dd, J = 9.5 Hz, Jꢀ = 4.9 Hz, 1H), 3.98 (m, 1H), 3.85
(m, 1H), 2.93 (t, J = 7.3 Hz, 2H), 2.53 (t, J = 7.4 Hz,
2H), 2.30 (broad, 1H), 1.63 (m, 4H), 1.32 (m, 6H), 0.93
(t, J = 7.3 Hz, 3H), 0.89 (t, J = 6.9 Hz, 3H); 13C NMR:
198.9, 160.5, 156.3, 136.0, 129.7, 114.4, 67.1, 62.5,
50.7, 37.3, 37.0, 31.7, 28.9, 24.9, 23.3, 22.6, 14.2, 13.9;
IR: 3590, 2932, 2396, 1738, 1684, 1510, 1373, 1216,
1119, 1072, 975 cm−1; [α]D = −3.81 (c = 1, CHCl3,
20 ◦C); HRMS—calculated for C20H31NO4 [M + Na]+:
372.2151; found: 372.2142.
Yield 63% after flash chromatography on 3:1 hex-
1
anes/ethyl acetate (Rf = 0.33); H NMR: 6.03 (broad,
1H), 5.36 (m, 2H), 4.06 (m, 1H), 3.84 (dd, J = 11.2 Hz,
Jꢀ = 5.0 Hz, 1H), 3.72 (dd, J = 11.3 Hz, Jꢀ = 3.5 Hz,
1H), 2.79 (dd, J = 13.6 Hz, Jꢀ = 6.5 Hz, 1H), 2.71 (dd,
J = 13.6 Hz, Jꢀ = 6.7 Hz, 1H), 2.59 (q, J = 7.4 Hz, 2H),
2.23 (t, J = 7.5 Hz, 2H), 2.02 (m, 2H), 1.64 (m, 2H),
1.32 (m, 17H), 0.89 (t, J = 6.7 Hz, 3H); 13C NMR:
173.8, 129.9, 129.6, 64.4, 50.6, 36.6, 32.5, 31.7, 29.7,
29.6, 29.6, 29.4, 29.2, 29.1, 29.0, 28.9, 28.8, 27.1, 27.0,
26.2, 25.5, 22.5, 14.6, 14.0; IR: 3371, 2924, 2855,
2382, 1652, 1551, 1463, 1371 cm−1; [α]D = +2.0 (c = 1,
CHCl3, 20 ◦C); HRMS—calculated for C23H45NO2S
[M + Na]+: 422.3069; found: 422.3060.
2.5.10. (2ꢀS)-N-(1-(4-(Decyloxy)phenoxy)-3-
hydroxypropan-2-yl)-2-oxooctanamide (10)
2.5.13. (2ꢀS,9E)-N-(1-Hydroxy-3-
(pentylthio)propan-2-yl)octadec-9-enamide (17)
Yield 61% after flash chromatography on 3:1 hex-
Yield 71% after flash chromatography on 3:1 hex-
anes/acetone; mp 87–88 ◦C; (Rf = 0.29); 1H NMR
anes/ethyl acetate (Rf = 0.31); H NMR: 6.06 (broad,
1

S. Grijalvo et al. / Chemistry and Physics of Lipids 144 (2006) 69–84
77
3.76 (dd, J = 11.1 Hz, Jꢀ = 4.5 Hz, 1H), 3.70 (s, 2H), 3.66
(dd, J = 11.1 Hz, Jꢀ = 3.8 Hz, 1H), 2.64 (dd, J = 13.6 Hz,
Jꢀ = 6.8 Hz, 1H), 2.60 (dd, J = 13.6 Hz, Jꢀ = 6.6 Hz, 1H),
2.18 (t, J = 7.5 Hz, 2H), 2.01 (m, 2H), 1.62 (m, 2H),
1.30 (m, 14H), 0.89 (t, J = 6.8 Hz, 3H); 13C NMR:
174.0, 136.7, 133.2, 130.5, 129.9, 63.9, 50.4, 37.0, 35.8,
32.3, 32.1, 29.9, 29.9, 29.7, 29.5, 29.5, 29.4, 29.3, 27.4,
27.4, 25.9, 22.9, 14.3; IR: 3300, 2927, 2853, 2361,
1643, 1544, 1494, 100, 1019 cm−1; [α]D = +7.7 (c = 1,
CHCl3, 20 ◦C); HRMS—calculated for C28H46ClNO2S
[M + Na]+: 518.2836; found: 518.2819.
1H), 5.34 (m, 2H), 4.03 (m, 1H), 3.80 (dd, J = 11.0 Hz,
Jꢀ = 4.7 Hz, 1H), 3.69 (dd, J = 11.4 Hz, Jꢀ = 3.6 Hz, 1H),
3.02 (broad, 1H), 2.75 (dd, J = 13.5 Hz, J’ = 6.5 Hz,
1H), 2.67 (dd, J = 13.5 Hz, Jꢀ = 6.7 Hz, 1H), 2.53 (t,
J = 7.3 Hz, 2H), 2.21 (t, J = 7.3 Hz, 2H), 2.00 (m, 2H),
1.60 (m, 4H), 1.28 (m, 18H), 0.88 (m, 6H); 13C
NMR: 173.8, 129.9, 129.6, 64.3, 50.6, 36.6, 32.9, 32.3,
31.8, 30.8, 29.6, 29.6, 29.4, 29.2, 29.1, 29.1, 29.0,
27.1, 27.0, 25.5, 22.1, 14.0, 13.8; IR: 3305, 2925,
2856, 1646, 1542, 1463, 1035 cm−1; [α]D = +10.3 (c = 1,
CHCl3, 20 ◦C); HRMS—calculated for C26H51NO2S
[M + Na]+: 464.3539; found: 464.3557.
2.5.17. (2ꢀS,9E)-N-(1-Hydroxy-3-(2-
naphylthio)propan-2-yl)octadec-9-enamide (21)
2.5.14. (2ꢀS,9E)-N-(1-Hydroxy-3-
(decylthio)propan-2-yl)octadec-9-enamide (18)
Yield 58% after flash chromatography on 4:1 hex-
anes/ethyl acetate; mp 53 ◦C; (Rf = 0.29); 1H NMR
(500 MHz): 6.05 (broad, 1H), 5.34 (m, 2H), 4.03 (m,
1H), 3.80 (dd, J = 11.0 Hz, Jꢀ = 4.4 Hz, 1H), 3.69 (m,
1H), 2.99 (broad, 1H), 2.75 (dd, J = 13.6 Hz, Jꢀ = 6.6 Hz,
1H), 2.67 (dd, J = 13.5 Hz, Jꢀ = 6.8 Hz, 1H), 2.53 (t,
J = 7.3 Hz, 2H), 2.21 (t, J = 7.3 Hz, 2H), 2.00 (m, 2H),
Yield 66% after flash chromatography on 3:1 hex-
anes/ethyl acetate; mp 71–72 ◦C; (Rf = 0.33); 1H NMR:
7.90 (s, 1H), 7.79 (m, 3H), 7.48 (m, 3H), 6.04
(broad, 1H), 5.36 (m, 2H), 4.15 (m, 1H), 3.90 (dd,
J = 11.2 Hz, Jꢀ = 4.5 Hz, 1H), 3.73 (dd, J = 11.1 Hz,
Jꢀ = 3.8 Hz, 1H), 3.30 (dd, J = 13.9 Hz, Jꢀ = 6.2 Hz,
1H), 3.25 (dd, J = 13.9 Hz, Jꢀ = 6.8 Hz, 1H), 2.12
(t, J = 7.4 Hz, 2H), 2.02 (m, 2H), 1.56 (m, 2H),
1.28 (m, 14H), 0.89 (t, J = 6.8 Hz, 3H); 13C NMR:
174.0, 133.9, 132.9, 132.1, 130.2, 129.9, 128.9,
127.9, 127.5, 127.4, 127.3, 126.9, 126.1, 63.8, 51.1,
36.9, 34.4, 32.1, 30.0, 29.9, 29.7, 29.5, 29.4, 29.3,
27.4, 27.4, 25.8, 22.9, 14.3; IR: 3310, 2918, 2853,
1628, 1557, 1466, 812, 741 cm−1; [α]D = +22.3 (c = 1,
CHCl3, 20 ◦C); HRMS—calculated for C31H47NO2S
[M + Na]+: 520.3225; found: 520.3210.
1.63 (m, 4H), 1.29 (m, 26H), 0.87 (t, J = 6.5 Hz, 6H); 13
C
NMR (125 MHz): 173.8, 129.9, 129.6, 64.4, 50.6, 36.6,
33.0, 32.3, 31.8, 29.6, 29.6, 29.4, 29.2, 29.1, 29.1, 29.0,
28.7, 27.1, 27.0, 25.5, 22.5, 14.0; IR: 3401, 3311, 2922,
2848, 1636, 1534, 1461, 1078 cm−1; [α]D = +9.7 (c = 1,
CHCl3, 20 ◦C); HRMS—calculated for C31H61NO2S
[M + Na]+: 534.4321; found: 534.4335.
2.5.15. (2ꢀS,9E)-N-(1-Hydroxy-3-
(hexadecylthio)propan-2-yl)octadec-9-enamide (19)
Yield 60% after flash chromatography on 4:1 hex-
anes/ethyl acetate (Rf = 0.27); 1H NMR (500 MHz): 6.05
(broad, 1H), 5.35 (m, 2H), 4.02 (m, 1H), 3.80 (dd,
J = 11.5 Hz, Jꢀ = 5.4 Hz, 1H), 3.69 (m, 1H), 3.00 (broad,
1H), 2.75 (dd, J = 13.5 Hz, Jꢀ = 6.5 Hz, 1H), 2.67 (dd,
J = 13.5 Hz, Jꢀ = 6.7 Hz, 1H), 2.53 (t, J = 7.3 Hz, 2H),
2.21 (t, J = 7.4 Hz, 2H), 2.00 (m, 2H), 1.60 (m, 4H), 1.28
(m, 42H), 0.87 (t, J = 6.5 Hz, 6H); 13C NMR (125 MHz):
173.8, 129.9, 129.6, 64.3, 50.6, 36.6, 33.0, 32.4, 31.8,
31.8, 29.6, 29.5, 29.5, 29.4, 29.4, 29.2, 29.2, 29.1, 29.1,
29.0, 28.7, 27.1, 27.0, 25.5, 22.5, 14.0; IR: 3317, 2922,
2854, 1640, 1557, 1467, 1045 cm−1; [α]D = +5.6 (c = 1,
CHCl3, 20 ◦C); HRMS—calculated for C37H73NO2S
[M + Na]+: 618.5260; found: 618.5241.
2.5.18. (2ꢀS,9E)-N-(1-Hydroxy-3-(4-(2-
methoxyethyl)phenoxy)propan-2-yl)octadec-9-
enamide
(22)
Yield 53% after flash chromatography on 3:1 hex-
anes/ethyl acetate (Rf = 0.29); 1H NMR: 7.14 (d,
J = 8.4 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H), 6.16 (broad,
1H), 5.33 (m, 2H), 4.30 (m, 1H), 4.10 (dd, J = 9.8 Hz,
Jꢀ = 4.7 Hz, 1H), 4.07 (dd, J = 9.4 Hz, Jꢀ = 4.5 Hz, 1H),
3.93 (dd, J = 11.1 Hz, Jꢀ = 4.0 Hz, 1H), 3.78 (dd,
J = 11.7 Hz, Jꢀ = 5.3 Hz, 1H), 3.55 (t, J = 7.0 Hz, 2H),
3.34 (s, 3H), 2.82 (t, J = 6.9 Hz, 2H), 2.35 (t, J = 7.4 Hz,
2H), 2.22 (t, J = 7.9 Hz, 2H), 2.00 (m, 2H), 1.63 (m,
2H), 1.27 (m, 12H), 0.87 (t, J = 6.4 Hz, 3H); 13C NMR:
178.4, 163.5, 135.3, 130.1, 114.6, 73.9, 63.5, 58.9, 50.6,
36.9, 35.5, 33.4, 32.1, 29.9, 29.9, 29.7, 29.5, 29.4, 29.3,
29.0, 27.4, 25.8, 22.9, 14.3; IR: 3310, 2922, 2851, 2383,
2304, 1659, 1513, 1464, 1243 cm−1; [α]D = −2.4 (c = 1,
CHCl3, 20 ◦C); HRMS—calculated for C30H51NO4
[M + Na]+: 512.3716; found: 512.3726.
2.5.16. (2ꢀS,9E)-N-(1-(4-Chlorobenzylthio)-3-
hydroxypropan-2-yl)octadec-9-enamide (20)
Yield 66% after flash chromatography on 3:1 hex-
anes/ethyl acetate; mp 48 ◦C; (Rf = 0.31); 1H NMR: 7.30
(m, 4H), 5.98 (broad, 1H), 5.36 (m, 2H), 4.06 (m, 1H),

78
S. Grijalvo et al. / Chemistry and Physics of Lipids 144 (2006) 69–84
2.5.19. (2ꢀS,9E)-N-(1-(4-Chloro-3-methylphenoxy)-
3-hydroxypropan-2-yl)octadec-9-enamide (23)
32.1, 29.9, 29.9, 29.8, 29.7, 29.7, 29.6, 29.5, 29.5,
29.4, 29.4, 29.3, 27.4, 27.3, 26.2, 25.9, 22.9, 14.3;
IR: 3323, 2953, 2921, 2851, 1642, 1562, 1508, 1460,
1240, 1051, 1016 cm−1; [α]D = −10.1 (c = 1, CHCl3,
20 ◦C); HRMS—calculated for C37H65NO4 [M + Na]+:
610.4811; found: 610.4832.
Yield 53% after flash chromatography on 3:1 hex-
anes/ethyl acetate (Rf = 0.28); 1H NMR: 7.23 (d,
J = 8.7 Hz, 1H), 6.79 (d, J = 2.9 Hz, 1H), 6.69 (dd,
J = 8.7 Hz, Jꢀ = 3.0 Hz, 1H), 6.07 (d, 1H), 5.34 (m,
2H), 4.27 (m, 1H), 4.12 (dd, J = 9.5 Hz, Jꢀ = 4.2 Hz,
1H), 4.06 (dd, J = 9.5 Hz, Jꢀ = 5.0 Hz, 1H), 3.93 (dd,
J = 11.2 Hz, Jꢀ = 4.4 Hz, 1H), 3.77 (dd, J = 10.9 Hz,
Jꢀ = 4.3 Hz, 1H), 2.59 (broad, 1H), 2.33 (s, 3H), 2.22
(t, J = 7.3 Hz, 2H), 2.00 (m, 2H), 1.62 (m, 2H),
1.26 (m, 14H), 0.87 (t, J = 6.5 Hz, 3H); 13C NMR:
173.6, 156.6, 137.1, 129.9, 129.6, 129.6, 126.4, 116.9,
112.9, 67.4, 62.7, 50.2, 36.6, 31.8, 29.6, 29.6, 29.4,
29.2, 29.1, 29.1, 29.0, 27.1, 27.0, 25.5, 22.5, 20.2,
14.0; IR: 3311, 2928, 2851, 2363, 2339, 1621, 1547,
1486, 1462, 1241, 1079 cm−1; [α]D = −6.9 (c = 1,
CHCl3, 20 ◦C); HRMS—calculated for C28H46ClNO3
[M + Na]+: 502.3064; found: 502.3045.
2.6. Biological activity
2.6.1. Inhibition of ceramidases in vitro
Mitochondria and lysosomes were used as the
nCDase and aCDase sources, respectively, and were pre-
pared from rat liver as reported (El Bawab et al., 2002;
Bedia et al., 2005). The effect of the compounds on
the aCDase activity was determined in 96-well plates
using a novel ceramide analog as substrate (Bedia et al.,
submitted for publication). To each well, the following
was added: 20 mM AcOH/AcONa buffer pH 4.5 contain-
ing0.25%TritonX-100(43 ␮l), testcompound(1.2 ␮lof
a 2.5 mM solution in ethanol, 3 nmol), substrate (1.2 ␮l
of a 2.5 mM solution in ethanol, 3 nmol) and protein
suspension (30 ␮l/well, 5 ␮g/␮l, 150 ␮g/well). After 3 h
at 37 ◦C, the reactions were stopped with methanol
(75 ␮l/well) and 100 mM glycine–NaOH buffer pH 10.6
(30 ␮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.
Enzyme activities were determined by comparison of
the experimental fluorescence values with a calibra-
tion curve prepared with umbelliferone, at final con-
centrations in the range of 0–3.2 ␮M. The effect of
the compounds on the nCDase activity were deter-
mined following the same procedure, but using 25 mM
Tris–HCl buffer pH 7.4, 0.1 mg of mitochondrial pro-
tein and a 1 h incubation period. Reaction was stopped
with 75 ␮l/well of methanol (no glycine buffer was
added).
2.5.20. (2ꢀS,9E)-N-(1-Hydroxy-3-(4-propylphenoxy)
propan-2-yl)octadec-9-enamide (24)
Yield 64% after flash chromatography on 3:1 hex-
anes/ethyl acetate; mp 110–111 ◦C; (Rf = 0.30); 1H
NMR: 7.08 (d, J = 8.7 Hz, 2H), 6.81 (d, J = 8.7 Hz, 2H),
6.22 (broad, 1H), 5.34 (m, 2H), 4.28 (m, 1H), 4.13
(dd, J = 9.6 Hz, Jꢀ = 4.1 Hz, 1H), 4.07 (dd, J = 9.5 Hz,
Jꢀ = 4.5 Hz, 1H), 3.93 (dd, J = 11.3 Hz, Jꢀ = 4.5 Hz, 1H),
3.78 (dd, J = 11.3 Hz, Jꢀ = 4.7 Hz, 1H), 2.52 (t, J = 7.4 Hz,
2H), 2.22 (t, J = 7.4 Hz, 2H), 1.99 (m, 2H), 1.60 (m,
2H), 1.27 (m, 22H), 0.91 (t, J = 7.3 Hz, 3H), 0.87 (t,
J = 6.9 Hz, 3H); 13C NMR: 174.4, 156.4, 136.0, 129.7,
126.7, 114.4, 112.1, 67.8, 63.4, 50.7, 37.3, 36.9, 32.1,
30.0, 29.9, 29.7, 29.5, 29.4, 29.4, 29.3, 27.4, 27.3, 25.9,
24.9, 22.9, 14.3, 13.9; IR: 3305, 2924, 2853, 2357,
1655, 1503, 1235 cm−1; [α]D = −3.9 (c = 1, CHCl3,
20 ◦C); HRMS—calculated for C30H51NO3 [M + Na]+:
496.3767; found: 496.3756.
2.5.21. (2ꢀS,9E)-N-(1-(4-(Decyloxy)phenoxy)-3-
hydroxypropan-2-yl)octadec-9-enamide (25)
Yield 67% after flash chromatography on 3:1 hex-
2.6.2. Inhibition of aCDase in cell culture
Fibroblasts from a Farber patient (Moh. pAS cell
line) transformed to overexpress the aCDase were
used. The cells were routinely grown in a humidified
5% CO2 atmosphere at 37 ◦C in Dulbecco’s modified
Eagel medium containing l-glutamine (2 mmol/l), peni-
cillin (100 U/ml), streptomycin (100 ␮g/ml) and heat-
inactivated fetal calf serum (10%). Twenty-four hours
before the experiment, cells were seeded (1/3 dilution
from a confluent culture in a 25 cm2 flask) in 96-well
plates (100 ␮l cell suspension/well). The medium was
1
anes/ethyl acetate (Rf = 0.32); mp 95–96 ◦C; H NMR:
6.82 (m, 4H), 6.16 (broad, 1H), 5.34 (m, 2H), 4.25
(m, 1H), 4.09 (dd, J = 9.5 Hz, Jꢀ = 4.1 Hz, 1H), 4.04
(dd, J = 9.6 Hz, Jꢀ = 4.7 Hz, 1H), 3.93 (m, 1H), 3.88 (t,
J = 6.6 Hz, 2H), 3.77 (m, 1H), 2.87 (broad, 1H), 2.21
(t, J = 7.3 Hz, 2H), 2.00 (m, 2H), 1.74 (q, J = 6.8 Hz,
2H), 1.63 (m, 2H), 1.43 (m, 2H), 1.28 (m, 26H), 0.87
(t, J = 6.7 Hz, 6H); 13C NMR: 174.0, 154.0, 152.3,
130.2, 129.9, 115.6, 115.6, 68.8, 68.4, 63.3, 50.7, 36.9,

S. Grijalvo et al. / Chemistry and Physics of Lipids 144 (2006) 69–84
79
removed and 100 ␮l of the same medium containing
16 ␮M of both the novel substrate and test compound
(substrate only in controls) were added. The plates were
placed back in the incubator and, after 4 h, methanol
(50 ␮l/well), NaIO4 (100 ␮l/well, 10 mg/ml in 0.1 M
phosphate buffer pH 8.0) and BSA (50 ␮l/well, 2 mg/ml
in 0.1 M phosphate buffer pH 8.0) were sequentially
added. The fluorescence was measured after 16 h in the
dark as indicated above.
submitted to coupling with the required carboxylic acid.
Amides 12–15 were obtained from direct coupling of
2-oxooctanoic acid or oleic acid with the corresponding
sphingoid base, which was either commercially avail-
able or had been obtained previously in our laboratories
(Triola et al., 2003).
3.2. Library diversity retrospective assessment
The design of a chemical library implies the proper
choice of building blocks in order to guarantee the maxi-
mum diversity of the library members. This is especially
important when small libraries are considered for pre-
liminary exploratory studies.
3. Results
3.1. Synthesis
Compounds 1–11 and 16–25 were synthesized from
tosylate 26 (Scheme 1), obtained by a slight modification
of a reported procedure (Garner and Park, 1987; Porte
et al., 1998). Nucleophilic displacement of the tosyloxy
group in 26 by a series of selected thiolates or alkox-
ides afforded the corresponding intermediates 27–37
in excellent yields. Acetonide hydrolysis and N-Boc
removal was carried out simultaneously by treatment
with a solution of trifluoroacetic acid in CH2Cl2 to fur-
nish the corresponding trifluoroacetate salts, which were
In order to assess the chemical diversity covered by
the synthesized combinatorial subset, a virtual combina-
torial library of 250 compounds was enumerated and the
coverage of the synthesized full-array was compared to
the best-optimized full-array selection.
This virtual combinatorial library is a two-component
library resulting from the derivatization of scaffold A
(Scheme 2) using two acyl groups (N-(2-oxoamide) and
N-oleoyl) for the first diversity point and 125 R groups
for the second diversity point. This last set of virtual
Scheme 1. (a) ROH or RSH/NaH/DMF, 40 ◦C, 1 h; (b) TFA/CH2Cl2, 5 min; (c) 1: RCOOH, HOBt-EDC/CH2Cl2; 2: Amberlite A-21, CH2Cl2.
Scheme 2. In silico design and analysis of the combinatorial library used in this study.

80
S. Grijalvo et al. / Chemistry and Physics of Lipids 144 (2006) 69–84
reactants consists of 91 phenols and 34 thiols, which
were selected by filtering a set of compounds extracted
from standard chemical product catalogues with drug-
likeness criteria. Obviously, the corresponding 11 R
groups (6 thioethers and 5 ethers) included in the 2 × 11
synthesized combinatorial subset, compounds (1–11 and
16–25), were also present in this list of 125 virtual reac-
tants.
metric procedure recently developed in our laborato-
ries, which is of use both in vitro and in cultured cells
(Bedia et al., submitted for publication). This assay
is based on the method developed by Reymond and
co-workers (Badalassi et al., 2000) and uses a novel
ceramide analog as substrate. The free base arising
from ceramidase hydrolysis is chemically oxidized with
NaIO4 to give an aldehyde that releases a highly fluo-
rescent umbelliferone upon ␤-elimination. Fluorescence
intensity is thus proportional to the enzyme hydrolytic
activity. This fluorogenic substrate is hydrolyzed by both
aCDase and nCDase with similar efficiencies, but it
is not a substrate of either anandamide hydrolase or
acid N-palmitoylethanolamine hydrolase (Bedia et al.,
submitted for publication).
Using this procedure, the activity of the compounds
synthesized as aCDase inhibitors was first investigated
in vitro (Fig. 2). In rat liver preparations incubated with
equimolar concentrations of substrate and test com-
pound (40 ␮M), ␣-ketoamides 1, 2, 4, 8 and 10 had
a slight, but statistically significant inhibitory activity
(around 20% inhibition). On the other hand, the N-2-
oxooctanamides of sphingosine (12), sphinganine (15)
and the acetylenic base (14) brought about a 25–50%
inhibition of the aCDase. Compound 12 seemed to be
the most potent, whereas its (Z)-isomer 13 was inactive
in the same experimental conditions. On the other hand,
neither NOE nor the N-oleoylethanolamine derivatives
16–25 inhibited aCDase activity in vitro. Finally none
of the above amides inhibited the nCDase at equimo-
lar concentrations (40 ␮M) with respect to the substrate,
whereas 40 ␮M sphingosine, which was used as a posi-
tive control (Usta et al., 2001), caused a 64% inhibition
(data not shown).
AfterenumerationwithMOEprogram(2004), energy
minimization was carried out with force field MMFF94
with0.01 kcal/molgradientforconvergence. Asetof100
standard 2D and 3D descriptors was calculated, among
them physical-chemistry, spatial, topological indices and
information content indices are included. Finally, dimen-
sionality of the data was reduced by principal com-
ponent analysis (PCA) down to eight components that
account for 90% of the variance. Diversity assessment
was measured by cell-based methods, in terms of both
space coverage (number of cells covered by selected sub-
set/number of total cells) and population coverage (total
number of compounds included in represented cells/total
number of molecules in library). In order to partition the
space, ward clustering was chosen as it yields homoge-
nous groups. Hierarchical tree level was stopped at
22 clusters that match the selection size. On the other
hand, simulated annealing was employed to optimize a
full-array selection recruited at the same ward-clustered
space.
Hence, the synthesized subset represents up to 60%
of cell coverage (65% population coverage), compared
to the maximum full-array optimal value found of 84%
(94% population). PRALINS, Program for Rational
Analysis of Libraries in silico (Pascual et al., 2003) car-
ried out the diversity selection and evaluation.
Using the same substrate and procedure, the activity
of the compounds was then investigated in cell culture
using fibroblasts from a Farber disease patient (Moh.
pAS cell line) transduced to overexpress aCDase. Pre-
liminary experiments evidenced that the fluorogenic sub-
3.3. Inhibition of ceramidases
The screening of the compounds as ceramidase
inhibitors was carried out in 96-well plates using a fluori-
Fig. 2. Effect of compounds on aCDase in vitro. The experiments were performed as described in Section 2. Bars represent the mean S.D. of three
to six replicates. Grey bars indicate statistically significant differences with respect to controls (unpaired two-tailed t-test, *p ≤ 0.05; **p ≤ 0.0005;
black bars, not significant).

S. Grijalvo et al. / Chemistry and Physics of Lipids 144 (2006) 69–84
81
Fig. 3. Effect of compounds on aCDase in cultured Farber fibroblasts (Moh. pAS cell line) overexpressing the aCDase. The experiments were
performed as described in Section 2. Bars represent the mean S.D. of three replicates. Grey bars indicate statistically significant differences with
respect to controls (unpaired two-tailed t-test, *p ≤ 0.05; **p ≤ 0.001; black bars, not significant). Compounds 10 and 13 were cytotoxic in the assay
conditions and their activity on aCDase could not be determined.
strate was not cytotoxic at concentrations up to 32 ␮M
for 24 h. Likewise, no cytotoxicity was produced by
the analogs (20 ␮M, 16 h), except for compounds 10
and 13, which decreased cell viability to 94% and
42%, respectively. Amongst the non-toxic compounds,
2-oxooctanamides 1–11 did not inhibit the aCDase activ-
ity in cell culture, whereas this enzyme activity was
reduced to approximately 40% with respect to controls
in the presence of the ␣-ketoamides of sphingosine (12),
sphinganine (15) and the acetylenic base (14) (Fig. 3).
On the other hand, very low, but significant inhibitory
activity was elicited by the N-oleoyl subset, except for
thioethers18and19, havingaC10andC16straightchain
substituent at the sulphur atom, respectively, and NOE,
which had no effect.
The common scaffold consisted on a simplified sph-
ingoid base moiety devoid of the C3 stereogenic cen-
tre and either a sulphur or an oxygen atom bridge,
which was introduced as a –CH2– bioisosteric unit to
ease the preparation by parallel synthetic methodol-
ogy. The skeletal variations on the sphingoid backbone
were properly chosen in order to maximize diversity as
found with PRALINS. Finally, the 2-oxooctanoyl group
was selected to discriminate between the aCDase and
nCDase (Bedia et al., 2005), since the latter requires
longer chain N-acyl groups for recognition (Usta et al.,
2001). Amongst the resultant N-2-oxooctanoyl deriva-
tives, compounds 1, 2, 4, 8 and 10 inhibited the aCDase
in vitro (Fig. 2), although with low potency. However,
no compound was inhibitory in cultured cells (Fig. 3), in
which 10, a moderate aCDase inhibitor in vitro, exhib-
ited a remarkable cytotoxicity. These results, compared
to the potency of the previously reported ␣-ketoamides
(Bedia et al., 2005), suggested that the sphingoid base
C3–OH was necessary for enhanced inhibitory potency.
To confirm this issue, the 2-oxooctanamides of sphingo-
sine (12), its (Z)-isomer (13), sphinganine (15), and the
4,5-acetylenic long chain base (14) were synthesized and
their activities were investigated. Compounds 12, 14 and
15 inhibited the aCDase both in vitro and in cultured cells
with higher effectiveness than compounds 1–11. Inter-
estingly, the 2-oxooctanamide of (Z)-sphingosine (13)
was not active in vitro (Fig. 2), which suggests that the
Z geometry of the sphingoid base double bond lowers
the affinity of the compound for the enzyme. This com-
pound exhibited a remarkable cytotoxicity and its effect
on aCDase in cultured cells could not be determined.
The mechanisms underlying the toxicity of both 13 and
10 are currently under investigation. In the case of 10,
it is possible that an accumulation of ceramide because
of aCDase inhibition will contribute to cell death. This
does not explain the toxicity of 13, since it did not inhibit
either aCDase or nCDase.
4. Discussion
In the light of their therapeutic potential, the search
for potent and selective aCDase inhibitors is of inter-
est. In a previous article (Bedia et al., 2005) we reported
on the aCDase inhibitory activity of ␣-ketoamides of
a sphingosine-like long chain base. This result was
not unexpected, since aCDase is a cysteine hydrolase
(Tsuboi et al., 2005) and several authors have reported
that ␣-ketoamides are active-site directed inhibitors of
serine and cysteine hydrolases, including fatty acid
amide hydrolases (Koutek et al., 1994; De Petrocellis
et al., 1997; Deutsch et al., 1997; Bisogno et al.,
1998; Boger et al., 1999, 2000; Vandevoorde et al.,
2003). Inhibition by these compounds occurs by reac-
tion of the electron deficient ␣-carbonyl group with the
active-site nucleophilic amino acid to form an hemiac-
etal intermediate, which mimics the reaction transition
state.
Since the ␣-ketoamide function appeared to be a
promising moiety leading to aCDase inhibitors, the syn-
thesis of a small ␣-ketoamide library was undertaken.

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S. Grijalvo et al. / Chemistry and Physics of Lipids 144 (2006) 69–84
Although nCDase is a serine protease (Galadari et
derivatives were above the critical micelle concentra-
tions. Another possible explanation is that inhibition is
not caused by the oleamides, but by the corresponding
amines resulting from hydrolysis mediated by fatty acid
amide hydrolases. However, the results obtained in the
experiments with amines putatively formed upon hydrol-
ysis of oleamides 17 and 19–22 do not support this
hypothesis. Thus, only the free base corresponding to 19,
which was not inhibitory in cells, was slightly inhibitory
in vitro (mean % of control S.D.: 78.0 6.0; signifi-
cantly different at p ≤ 0.005, unpaired two-tailed t-test)
whereas the other amines, corresponding to oleamides
effective in cells (17 and 20–22), did not inhibit the
aCDase in vitro (data not shown). Finally, it is also pos-
sible that the observed inhibition by oleamides in intact
cells is secondary to the interaction of the compounds
with their real intracellular target.
al., 2005) and therefore, potentially sensitive to ␣-
ketoamides, none of the compounds 1–15 inhibited this
enzyme. As discussed in a previous article (Bedia et
al., 2005), assuming that ceramidase inhibition by ␣-
ketoamides occurs at the enzyme active centre (Koutek
et al., 1994; De Petrocellis et al., 1997; Deutsch et al.,
1997; Bisogno et al., 1998; Boger et al., 1999, 2000;
Vandevoorde et al., 2003), and considering the structural
requirement of nCDase for substrates with long chain
N-acyl groups (Usta et al., 2001), the ␣-oxooctanamide
unit is too short for the appropriate binding needed for
inhibition. Besides the presence of the N-2-oxooctanoyl
group, the absence of the C3–OH function in compounds
1–11 may further reduce their enzyme affinity, since this
secondary hydroxyl group is also important for nCDase
activity (Usta et al., 2001). Additional affinity decrease
may be also involved in the lack of effect of compounds
13–15, since the presence of an (E)-4,5 double bond in
the substrate is required for efficient nCDase hydrolysis
(Usta et al., 2001).
As mentioned in Section 1, NOE has been reported
as an inhibitor of aCDase. Nevertheless, it is quite weak,
unselective, and inhibition in cell culture does not seem
reproducible. Since N-oleoylsphingosine is an aCDase
substrate (Al et al., 1989), it appears that removal of
the 3-hydroxy-4,5-hexadecenyl tail converts a substrate
into a poor inhibitor. Thus, we envisaged that better
aCDase inhibitors might be obtained if the tail is not
removed, but structurally modified. To explore this pos-
sibility, the same amine precursors of the ␣-ketoamide
library were N-acylated with oleic acid to obtain a family
of NOE analogues substituted at C2. Neither NOE nor
any of its analogs inhibited the aCDase or the nCDase
activity in vitro. Likewise, NOE, 18 and 19 were not
active on aCDase in cell culture. Conversely, all the
other compounds of this series inhibited the aCDase
in cultured cells, although with a rather low but sig-
nificant potency. The lack of activity of NOE, 18 and
19 in the cell model used here may indicate that these
compounds do not have the needed hydrophobicity for
activity (i.e. to cross the cell membrane). Accordingly,
while the estimated C log P of the active compounds
lies within the range 8.5–10.5, that of NOE is two units
lower (C log P 6.57) and those of 18 and 19, having the
longer chains at C2, are around 2 and 5 units higher,
respectively (C log P: 18, 12.66; 19, 15.83). The dif-
ferent activity exhibited by the N-oleoylethanolamine
family in vitro and in cell culture may indicate that inhi-
bition in vitro requires higher concentration than in cul-
tured cells. Unfortunately, this hypothesis could not be
confirmed, since higher concentrations of the N-oleoyl
The ineffectiveness of these compounds on the
nCDase can be explained, as mentioned above for the
␣-ketoamides, invoking a lack of affinity for the enzyme
arising from the absence of the C3–OH group. Never-
theless, this explanation is valid for compounds acting
on the enzyme active-site, which is not known for NOE.
5. Concluding remarks
Acylation of a sphingoid base amino group with elec-
tron deficient-containing moieties, such as 2-oxoacyl
groups, appears to be a suitable strategy to obtain cerami-
dase inhibitors. The use of short chain acylating units
confers specificity for the aCDase in front of the neutral
enzyme. Although the C3–OH group seems to be neces-
sary for inhibition, the double bond at C4 is not a strict
requirement for inhibitory activity. On the other hand,
chemical modification of the N-oleoylsphingosine back-
bone does not seem to be an appropriate approach to find
aCDase inhibitors.
Acknowledgements
Partial financial support from Ministerio de Cien-
´
cia y Tecnologıa (Spain) (Projects MCYT BQU2002-
03737 and CTQ2005-00175/BQU) and Fondos Feder
´
´
´
´
(EU), Fundacion Ramon Areces, Fundacio La Marato de
TV3 (Projects 040730 and 040731) and Generalitat de
Catalunya (Projects 2005SGR01063) is acknowledged.
CB, SG, and OR are also acknowledged to Ministerio
´
de Educacion y Ciencia (Spain), CSIC, and Generalitat
de Catalunya, respectively, for predoctoral fellowships;
GT thanks Genzyme for partial support of a CSIC-I3P
contract.

S. Grijalvo et al. / Chemistry and Physics of Lipids 144 (2006) 69–84
83
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