Synthesis and biological properties of Pachastrissamine (jaspine B) and diastereoisomeric jaspines

Article abstract of DOI:10.1016/j.bmc.2008.11.026

The synthesis of isomeric jaspines (anhydro phytosphingosines), arising from intramolecular cyclization of the corresponding phytosphingosines with different configurations at C3 and C4 positions of the sphingoid backbone, is reported. Natural jaspine B i

Full text of DOI:10.1016/j.bmc.2008.11.026

Bioorganic & Medicinal Chemistry 17 (2009) 235–241
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological properties of Pachastrissamine (jaspine B)
and diastereoisomeric jaspines
Daniel Canals a, , David Mormeneo a,b, , Gemma Fabriàs a, Amadeu Llebaria a,
a,b,
Josefina Casas a, , Antonio Delgado
*
*
a Research Unit on BioActive Molecules (RUBAM), Departament de Química Biomèdica, Institut de Química Avançada de Catalunya (IQAC-C.S.I.C),
Jordi Girona 18-26, 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 synthesis of isomeric jaspines (anhydro phytosphingosines), arising from intramolecular cyclization
of the corresponding phytosphingosines with different configurations at C3 and C4 positions of the
sphingoid backbone, is reported. Natural jaspine B is the most cytotoxic isomer on A549 cells and it
induces cell death in a dose-dependent manner. The cytotoxicity of jaspine B has been correlated with
a significant increase of intracellular dihydroceramides, which seem to play an active role in autophagy.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 29 September 2008
Revised 30 October 2008
Accepted 3 November 2008
Available online 18 November 2008
Keywords:
Phytosphingosine
Dihydroceramide
Ceramide
Cytotoxicity
Stereoisomers
1. Introduction
CH3
O
( )12
CH3
CH3
O
O
( )12
( )12
Sphingolipids (SLs) comprise ubiquitous natural products with
essential roles in cell biology. From a chemical perspective, mam-
malian SLs derive mainly from (E,2S,3R)-2-amino-4-octadecen-1,3-
diol or sphingosine (Sph), although a minor portion arise from the
saturated analogue sphinganine or its 4R-hydroxy derivative
phytosphingosine.
5
2
5
2
HN
O
4
3
4
3
H2N
OH
H2N
OH
H
Jaspine B
(2S,3S,4S)
2-epi-Jaspine B
(2 ,3 ,4
OH
Jaspine A
R
S S)
Some unusual sphingolipids exhibiting potent antitumor activ-
ity have been described from marine organisms.1 In 2002, Kuroda
and co-workers reported on the isolation and characterization of
Pachastrissamine, an anhydro phytosphingosine isolated from the
marine sponge Pachastrissa sp.2 This compound is also known as
jaspine B (Fig. 1) after its isolation from the sponge Jaspis sp.3–5
Other structurally related analogues have also been isolated from
the same species, including jaspine A and jaspine B,4 whose C2 epi-
mer (Fig. 1) has also been reported.5 This compound, initially de-
scribed as a plant metabolite,6 was first characterized by total
synthesis of a shorter analogue,7–9 and later reported as a chemical
entity, both in the form of free amine10–13 or as the corresponding
N-benzoyl amide in the course of a series of chemical transforma-
Figure 1. Structure of natural jaspines.
The structure of jaspine B has been unambiguously confirmed
by total synthesis from
tosphingosine,12,13 -xylose,16 (R)-glycidol,17
and a galactal derivative,19 among others.20,21
L D-ribo phy-
-serine,15 Garner’s aldehyde,11
D
D
-(ꢀ)-tartaric acid,18
Jaspine B shows cytotoxicity against several cell lines at a nano-
molar level.2,4 However, despite its remarkable biological activity,
little is known about the structure–activity relationships in jaspine
B and, in particular, on the role of the stereochemistry of the tetra-
hydrofuran substituents on the biological profile of this interesting
family of compounds. Based on these premises, we undertook the
synthesis and biological evaluation of all four jaspine stereoisomers
(2a–2d) arising from the intramolecular cyclization of N-Boc phy-
tosphingosines (1a–1d) with different configuration around the
C3 and C4 positions of the sphingoid backbone (Scheme 1).22 To
tions from N-substituted D
-ribo phytosphingosine derivatives.14
* Corresponding authors. Tel.: +34 934006108; fax: +34 932045904.
E-mail address: adelgado@cid.csic.es (A. Delgado).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.026

236
D. Canals et al. / Bioorg. Med. Chem. 17 (2009) 235–241
OH
OH
1
1
O
2
4
O
2
4
C14H29
C14H29
C14H29
C14H29
2
2
HO
Boc
HO
3
3
NH OH
NH OH
4
3
4
3
Boc
1a: D-ribo (2
H2N
OH
H2N
,3 ,4
OH
1c: D-xylo (2S,3R,4R)
2c (2R,3R,4S)
S
,3
S,4R)
2a (2
R
S
S
) (2-epi Jaspine B)
OH
OH
1
1
O
2
4
O
2
4
C14H29
C14H29
C14H29
C14H29
2
2
HO
3
HO
3
NH OH
NH OH
4
3
4
3
Boc
1b: L-arabino (2S,
Boc
1d: L-lyxo (2S,3S,4S)
H2N
2b (2S,
OH
,4
H2N
OH
3R,4S)
3
R
S)
2d (2S,3S,4S)(Jaspine B)
Scheme 1. Stereoisomeric N-Boc phytosphingosines and their corresponding anhydro derivatives (see also Ref. 4).
our knowledge, 2b and 2c are unprecedented, whereas no biological
data have been reported in the literature for 2a.
tivity of jaspine isomers in this assay, since the less toxic isomer
2c differs from jaspine B (2d) in both C3 and C4 stereocenters.
Interestingly, phytosphinogosines derived from N-Boc removal of
1a–1d were 7–140 times less toxic than the configurationally
equivalent cyclic analogues 2a–2d.25
Since the MTT test is a mere measure of living cells, the toxic ef-
fects of jaspine stereoisomers were investigated by flow cytometry
analysis after double labelling with annexin V (AV) and propidium
iodide (PI). The results on A549 cells cultured in the presence of
2a–2d at different concentrations are shown in Figure 2. In control
experiments, the percentage of AV-stained cells (2%) was not sig-
nificantly increased after treatment with jaspine B (2d) or stereo-
isomeric jaspines 2a–2c. However, the percentage of AV-PI
double positive cells increased moderately at high concentrations
2. Results and discussion
2.1. Chemistry
Jaspines 2a–2d were obtained by intramolecular cyclization of
N-Boc phytosphingosines (1a–1d), resulting from Sharpless asym-
metric dihydroxylation of the corresponding olefins.23 Treatment
of 1a–1c with p-toluenesulfonyl chloride in CH2Cl2/pyridine led
to 4a–4c, presumably through in situ cyclization of the transient
tosylates 3a–3c arising from selective tosylation of the primary
alcohol, as described in related systems (Scheme 2).24,12 However,
treatment of 1d under similar reaction conditions allowed the iso-
lation of tosylate 3d, which was further cyclised to 4d following re-
ported protocols.17
In all cases, deprotection of the N-Boc group in 4a–4d under
standard acidic conditions (TFA/CH2Cl2 5:1, rt, 1 h) led to the ex-
pected jaspines 2a–2d in good yields (74–82%). These were fully
characterized by NMR methods, by comparison with reported data,
for 2a5,10,12 and 2d,2,4,5,17,12,13 and also by 2D experiments for unre-
ported 2b and 2c.
of 2a–2c (1 and 2 lM), whereas this effect was less remarkable
for jaspine B (2d). Finally, a concentration-dependent increase in
the percentage of PI positive cells was observed in all cases
(Fig. 2). These results demonstrate that apoptosis only accounts
for a minor percentage of cell death and, therefore, a different cell
death mechanism must be implicated.
2.2.2. Cytotoxicity of compounds 2a–2d decreases in the
presence of 3-methyladenine
Since it has been recently demonstrated that some agents used
in antitumor therapy are able to induce autophagy, but not apop-
tosis, in several cancer cells,26 we explored the ability of the jaspine
analogues to promote this type of programmed cell death in A549
cells. Among the different described methods for monitoring
autophagy, inhibition of cell death by 3-methyladenine (3-MA) is
a frequently used proof to confirm the autophagy process.27,28 Un-
der our assay conditions, no significant differences (p P 0.05) were
observed between cells treated with 3-MA and those treated with
the vehicle only. Conversely, a marked decrease in toxicity of jas-
2.2. Biological results
2.2.1. Cytotoxic effects of 2a–2d on A549 cells
Cytotoxicity of jaspines 2a–2d was examined in A549 human
alveolar cells by the MTT test. Jaspine B (2d) was the most cyto-
toxic compound in this assay (LD50 = 0.13 0.01
stereomeric jaspines 2a–2c were about 10–20 times less toxic, as
judged by their LD50 values (2a, 1.25 0.03 M; 2b, 1.50
0.03 M; 2c, 2.5 0.04 M). These data point out the stereoselec-
lM), whereas dia-
l
l
l
H
O
O
C14H29
TsO
a
a
d
C14H29
(from 1a-c)
Boc-HN
OH
4a-4c
Boc-HN
OH
3a-3c
OH
C14H29
2a, 2b, 2c
2d
HO
NH OH
Boc
1a-1c
1d
OH
O
C14H29
C14H29
b
c
d
TsO
Boc
NH OH
Boc-HN
OH
3d
4d
Scheme 2. Reagents and conditions: (a) TsCl (1.1 equiv/mol), pyr/CH2Cl2 (1:1), 25 °C, 20 h; (b) TsCl (3 equiv/mol), TEA, cat DMAP, CH2Cl2; (c) K2CO3, MeOH, 20 h, 0–25 °C; (d)
TFA/CH2Cl2 5:1, rt, 1 h.

D. Canals et al. / Bioorg. Med. Chem. 17 (2009) 235–241
237
90
80
70
60
50
40
30
20
10
0
PI positive
Annexin positive-PI positive
Annexin positive
C
0.1 0.2 0.5
1
2
0.1 0.2 0.5
1
2
0.1 0.2 0.5
2c
1
2
0.03 0.06 0.1 0.2 0.5 (μM)
2a
2b
2d
Figure 2. Flow cytometry analysis of A549 control (C) and treated cells for 24 h at the indicated concentrations of compounds 2a–2d. Y axis indicates the percentage of cell
population stained with annexin V, propidium iodide or both.
2.2.3. Jaspine B modifies ceramide and dihydroceramide levels
in A549 cells
Recent studies have established that endogenous sphingolipids
are involved in autophagy. Lavieu et al.29 reported that ceramide
(Cer) and sphingosine-1-phosphate (Sph1P) are able to trigger
autophagy with opposing outcomes on cell survival. On the other
hand, an increase of endogenous dihydroceramides (DHCer’s)
was detected in cells treated with fenretinide,26 a compound that
has been reported to cause autophagy.30 In order to elucidate if
sphingolipids are involved in the 3-MA-inhibited cytotoxicity in-
duced by jaspine B, its effect on the ceramidome of A549 cells
was investigated.
The concentration-dependent effects of jaspine B on endoge-
nous Cer and DHCer levels at 10 h are shown in Figure 4 and the
time-course variation of Cer and DHCer amounts in A549 cells cul-
tured with 50 and 200 nM of jaspine B are shown in Figure 5. Cells
treated with 5 and 10 nM of jaspine B exhibited the same levels of
Cer’s and DHCer’s than controls. Regarding Cer levels, a 1.2- to 1.4-
fold increase in C16Cer was observed when A549 cells were treated
at concentrations P25 nM of jaspine B. Conversely, levels of
C24Cer and C24:1Cer remained unaffected up to 200 nM jaspine
B concentration, in which a slight but significant increase in both
species was found.
Jaspine B produced both a concentration and time-dependent
effect on the DHCer production. Thus, an increase of C24DHCer
and C24:1DHCer was already detected after 2 h incubation with
50 nM (a non-cytotoxic concentration) of jaspine B. After 10 h
incubation (Fig. 4), C24DHCer exhibited the highest build up as jas-
pine B concentrations raised, reaching a 2.5-fold increased at
200 nM. A similar trend was observed for C16DHCer, although only
Figure 3. (A) Cytotoxicity in A549 cells of 2d in the presence (full symbols) or in the
absence (empty symbols) of 10 mM 3-MA. (B) Cytotoxicity (LD50 values) of 2a–2d in
a 1.7-fold increase took place for this species at the same concen-
tration. Finally, levels of C24:1DHCer attained a 1.3-fold increase at
25 nM, which remained stable up to 100 nM.
A549 cells in the presence (full bars) or in the absence (empty bars) of 10 mM 3-MA.
Values are means SD of three independent experiments with triplicates.
The time-course increase in DHCer’s followed different profiles
depending on the molecular species (Fig. 5). Thus, both C24DHCer
and C24:1DHCer levels rose up after 10 h treatment (2.6- and 1.8-
fold increase at 200 nM, respectively), followed by a decrease at
24 h. However, C16DHCer levels remained increasing up to 24 h,
when a 2.7-fold increase at 200 nM occurred.
pine B in the presence of 3-MA was found (Fig. 3A). Thus, the LD50
value calculated for jaspine B in the presence of 3-MA increased
around 15 times in comparison with that found in the absence of
3-MA (Fig. 3B). Similar results, albeit less remarkable, were found
for compounds 2a–2c, where an increase in the LD50 values by a
factor of 2 (for 2a–2b) and 3 (for 2c) were observed. Interestingly,
the presumed autophagic activity profile shown by jaspine B seems
related to its cyclic nature, since the configurationally equivalent
3. Discussion
open chain
both in the absence or the presence of 3-MA (28.2 0.42 vs
29.4 0.54 M, p P 0.05). These results are indicative of a hitherto
unreported mode of action for jaspine B and its stereoisomers.
L-lyxo-phytosphingosine exhibited similar cytotoxicity
The marine environment is a source of extremely potent com-
pounds that have demonstrated, among many other properties,
significant biological activities as antitumor, and cytotoxic
l

238
D. Canals et al. / Bioorg. Med. Chem. 17 (2009) 235–241
300
250
200
150
100
C16DHCer
C16Cer
C24DHCer
C24Cer
C24:1DHCer
C24:1Cer
0
0.05
0.1
0.15
0.2
μ
Concentration ( M)
Figure 4. Concentration-dependent effects of jaspine B on endogenous ceramide (Cer) and dihydroceramide (DHCer) species at 10 h of treatment. C16, C24, and C24:1
indicate the N-acyl chain length. Data are means SD of three experiments with triplicates.
double staining with propidium iodide and annexin V (Fig. 2, 2d)
showed that less than 5% of the cells were annexin V positive
and around a 20% were annexin V positive/propidium iodide posi-
tive. These results demonstrate that apoptosis can only account for
a minor percentage of cell death and, therefore, most of the treated
cells must die by a different mechanism.
Autophagy, also named as type II programmed cell death, is a
dynamic process in which proteins and altered organelles are
sequestered inside cytoplasmic membrane structures for further
degradation and recycling. An increasing number of studies report
that autophagy is activated in cancer cells in response to various
anticancer therapies, such as tamoxifen in breast cancer cells,36
or fenretinide in prostate cancer cell lines.37 As for natural prod-
ucts, resveratrol, a phytoalexin that is present in grape nuts and
red wine, induced autophagy in ovarian cancer cells38 and soybean
B group triterpenoid saponins caused autophagy in colon cancer
cells.39 Genistein, an isoflavonoid abundant in soy products, in-
duced apoptosis and autophagy in a dose-dependent response.40
A similar dual effect has been reported for curcumin41 and for Sola-
num nigrum, an herbal plant whose extracts are used in traditional
oriental medicines.42 In the present study, an autophagy inhibitor
(3-MA) has been used to explore whether autophagy may play a
Figure 5. Time-course effects of jaspine B on endogenous ceramide (Cer) and
role in the cytotoxicity of jaspine B (2d) and that of the stereoiso-
dihydroceramide (DHCer) species at 50 nM (A) and 200 nM (B). C16, C24, and C24:1
meric jaspines 2a-2c. Although compound 3-MA inhibits class I
indicate the N-acyl chain length. Data are means SD of three experiments with
and class III phosphatidylinositol 3-kinases, two enzymes with op-
triplicates.
posed functions in autophagy, the overall effect of this inhibitor is
typically to block autophagy.27 As shown in Figure 3A, 500 nM jas-
drugs.31–33 In particular, marine sponges are the source of a wide
variety of sphingosine-related compounds.1 Among them, jaspine
B represents the first natural anhydro phytosphingosine reported
so far. It is cytotoxic against several cell lines at a nanomolar level
through a yet unknown mechanism.
The tetrahydrofuran backbone is an ubiquitous heterocyclic
unit found in a number of biologically active natural products.34
It is essential for the cytotoxic activity of jaspine B, since the re-
lated open-chain phyosphingosines were 10–100 times less toxic.
Nevertheless, a systematic survey of the biological stereoselectivity
of jaspine B isomers has not yet been reported. Our data show that
inversion of C2 and/or C3 stereocenters provides less toxic
compounds.
pine B caused a 95% cell death, whereas no cytotoxicity was de-
tected at the same concentration in the presence of 3-MA. A
similar, although less remarkable, cytoprotective effect of 3-MA
was observed for related stereoisomers (2a–2c).
Scarlatti et al.36 have established that Cer’s could mediate the
autophagy induced by tamoxifen in MCF-7 cells, whereas an in-
crease of endogenous DHCer’s, but not Cer’s, were observed in
DU145 cells treated with fenretinide, a compound that also causes
autophagy.30 In addition, exogenously added cell permeable Cer
can induce autophagy in malignant glioma cells43 and in HT-29
cells by increasing the intracellular pool of long chain Cer’s.36 On
the other hand, the effect of cell permeable DHCer seems to be cell
specific, since it has been reported to be ineffective toward autoph-
agy in HT-29,36 whereas Zheng et al. reported that exogenous
DHCer was able to induce autophagy in DU145 cells.30 Our results
in cultured A549 cells indicated that jaspine B caused a slight in-
crease in Cer contents with a substantial concentration-dependent
It has been reported that phytosphingosine induces apoptosis,
which is the most common mechanism of programmed cell death
in human cancer cells.35 When A549 cells were cultured with jas-
pine B at different concentrations, flow cytometry analysis after

D. Canals et al. / Bioorg. Med. Chem. 17 (2009) 235–241
239
and time-course-dependent effect on DHCer production. DHCer
0.125, 0.25, 0.5, 1, and 2
lM, whereas jaspine B (2d) at 0.033,
levels in A549 cells increased up to 3-fold after treatment with
200 nM jaspine B. Since DHCer’s have traditionally been consid-
ered as inert lipids, reports on variations of their intracellular levels
are scarce in the literature. Increases on DHCer levels have been re-
ported in different cell types after incubation with DHCer desatur-
0.066, 0.125, 0.25, and 0.5 lM.
5.3. LC–MS analysis of sphingolipids
A549 cells were seeded in 1 mL of medium with 10% FBS in a 6-
well plates. Twenty-four hours later, media were replaced with
fresh medium containing jaspine B at different concentrations (5,
10, 25, 50, 100, and 200 nM) and cultured for different times (1,
2, 10, and 24 h) at 37 °C/5% CO2. Then, cells were washed in PBS,
collected by brief trypsinization and 106 cells transferred to glass
vials. An aliquot of cells was taken for protein measurements.
Sphingolipid extracts, fortified with internal standards (N-dode-
canoylsphingosine, N-dodecanoylglucosylsphingosine and N-
dodecanoyl sphingosylphosphorylcholine, 0.5 nmol each), were
prepared as described30 and analysed. The liquid chromatogra-
phy–mass spectrometer consisted of a Waters Aquity UPLC system
connected to a Waters LCT Premier orthogonal accelerated time of
flight mass spectrometer (Waters, Millford, MA), operated in posi-
tive electrospray ionisation mode. Full scan spectra from 50 to
1500 Da were acquired and individual spectra were summed to
produce data points each 0.2 s. Mass accuracy and reproducibility
were maintained by using an independent reference spray via
ase inhibitors,44 fenretinide,37,26 or -tocopherol.45 These increases
c
were higher than those found in this work with jaspine B, although
the different cell models and compound used in the several studies
can account for these differences. Accumulation of DHCer suggests
a reduced DHCer desaturase activity upon treatments. While the
activity of fenretinide as a DHCer desaturase inhibitor has been
documented37,26 and Jiang et al. suggested that DHCer desaturase
is likely to be inhibited as a result of c-tocopherol treatment, nei-
ther jaspine B nor its stereoisomers 2a–c inhibit DHCer desatur-
ase.25 Whether the protein expression is downregulated by this
compound is under investigation.
4. Conclusion
In summary, we have demonstrated that jaspine B and its dia-
stereoisomers 2a–2c caused cell death in A549 cells in a dose-
dependent manner, and that this effect was prevented by 3-MA,
an autophagy inhibitor. Moreover, cell death was accompanied
the LockSpray interference. The analytical column was
a
by
a concentration- and time-course dependent build-up of
100 mm ꢁ 2.1mm id, 1.7
l
m C8 Acquity UPLC BEH (Waters). The
DHCer’s, but not Cer’s. Collectively, these results suggest that
DHCer-mediated autophagy might be involved in the cytotoxicity
of jaspine B. This possibility, along with the cell signalling path-
ways implicated will be investigated in the near future and will
be reported in due course.
two mobile phases were phase A: MeOH/H2O/HCOOH (74:25:1 v/
v/v); phase B: MeOH/HCOOH (99/1 v/v), both also contained
5 mM ammonium formate.
0.0 min, 80% B; 3 min, 90% B; 6 min, 90% B; 15 min, 99% B;
18 min, 99% B; 20 min, 80% B. The flow rate was 0.3 mL minꢀ1
A gradient was programmed—
.
The column was held at 30 °C. Quantification was carried out using
the extracted ion chromatogram of each compound, using 50 mDa
windows. The linear dynamic range was determined by injecting
standard mixtures. Positive identification of compounds was based
on the accurate mass measurement with an error <5 ppm and its
LC retention time, compared to that of a standard ( 2%).
5. Experimental
5.1. Cytotoxicity assay in A549 cells
Human alveolar epithelial A549 cells were obtained from the
American Type Culture Collection (ATCC) and grown in HAM F12
with glutamine medium supplemented with 10% foetal bovine ser-
um (FBS). Cells were kept at 37 °C in 5% CO2/95% air. At the time of
the experiments, cells were seeded in medium with 10% FBS at 105
cells per well in 96-well plates. Twenty-four hours later, media
were replaced with fresh medium and compounds were added to
give final concentrations of 0.02–400 lM. Cells were incubated at
37 °C in 5% CO2/95% air for 24 h. The number of viable cells was
quantified by the estimation of its dehydrogenase activity, which
5.4. Chemistry
Solvents were distilled prior to use and dried by standard meth-
ods. FT-IR spectra are reported in cmꢀ1 1H and 13CNMR spectra
.
were obtained in CDCl3 solutions at 300 MHz (for 1H) and
75 MHz (for 13C), respectively, unless otherwise indicated. Chemi-
cal shifts (d) are reported in ppm relative to the solvent (CDCl3) sig-
nal. ESI/HRMS spectra were recorded on a Waters LCT Premier
Mass spectrometer.
reduces
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) to water-insoluble formazan, which was dissolved
in DMSO and measured at 570 nm with a Multiskan plate reader
(Labsystems). All compounds were dissolved in ethanol and con-
trol experiments were performed with ethanol (0.1%). For suppres-
sion of autophagy, cells were treated with the inhibitor 3-
methyladenine (3-MA) at a concentration of 10 mM for 2 weeks46
on a 25 mL flask, changing the medium every 2 days. Then, 105
cells were seeded in 96-well plates in media with/without 3-MA
containing compounds 2a–2d at the above-mentioned concentra-
tions and cultured for 24 h. The number of viable cells was esti-
mated with the MTT test.
5.4.1. (20S,30S,40S)-[2-(tert-Butoxycarbonylamino)-3,4-dihydrox-
yoctadecyl]-p-toluenesulfonate (3d)
A suspension of triol 1d (250 mg, 0.60 mmol) in 20 mL of CH2Cl2
under Ar was cooled to 0 °C and treated with 0.45 mL (3.2 mmol) of
freshly distilled TEA, 6 mg of DMAP and 343 mg (1.8 mmol) of TsCl.
The reaction was stirred at 25 °C for 6 h, while the white suspen-
sion became slowly a clear solution. Solvents were removed at re-
duced pressure and the resulting residue was purified by flash
chromatography (Hexane/EtOAc 8:2) to afford 4d in 66% yield;
mp 63–64 °C; ½a 2D5
ꢀ3.4 (c 0.82, CHCl3); IR (film): 3381, 2923,
ꢂ
2853, 1684, 1522, 1456, 1366, 1288, 1252, 1175, 1096, 1051,
979. 1H NMR (CDCl3, 500 MHz): 7.79 (d, J = 8 Hz, 2H), 7.35 (d,
J = 8.5 Hz, 2H), 5.11 (d, J = 9 Hz, 1H, NH), 4.43 (dd, J = 10, 3.5 Hz,
1H, H1a), 4.23 (dd, J = 10, 3 Hz, 1H, H1b), 3.65 (m, 1H, H2), 3.54
(m, 1H, H3), 3.23 (d, J = 9.5 Hz, 1H, H4), 2.45 (s, 3H), 1.61 (m,
1H), 1.44 (s, 9H), 1.39 (m, 1H), 1.22–1.34 (m, 24H), 0.87 (t,
J = 6.7 Hz, 3H). 13C NMR (CDCl3, 100 MHz): 157.1, 145.4, 132.4,
130.2, 128.1, 81.1, 71.9, 69.9, 68.9, 51.8, 32.6, 32.0, 29.8, 29.8,
5.2. Flow cytometry analysis
A549 cells cultured with different concentration of compounds
2a–2d for 24 h. Cells were collected by brief trypsinization, stained
with an Alexa Fluor 488 annexin V/propidium iodide staining kit
(Molecular Probes, Inc. Oregon), and evaluated by FACS (Coulter
XL) with Coulter EPLDS.47 Compounds 2a–2c were tested at

240
D. Canals et al. / Bioorg. Med. Chem. 17 (2009) 235–241
29.7, 29.6, 29.5, 28.3, 26.2, 22.8, 21.8, 14.3. ESI-MS m/z 472.2
[MꢀBoc+H]+.
1.53 (m, 1H), 1.45 (s, 9H), 1.24–1.35 (m, 24H), 0.88 (t, J = 7 Hz,
3H). 13C NMR (CDCl3, 100 MHz): 156.0, 82.3, 80.0, 77.5, 72.0,
54.3, 32.1, 29.9, 29.8, 29.8, 29.8, 29.7, 29.7, 29.5, 29.1, 28.5, 26.2,
22.8, 14.3. ESI-MS m/z 422.3 [M+Na], 300.3 [MꢀBoc].
5.4.2. Synthesis of tetrahydrofuran derivatives 4a–4c from 1a
to 1c
General method. The corresponding N-Boc protected phyto-
sphingosine 1a–1c (0.20 mmol) and 41 mg (0.22 mmol) of TsCl
were weighted in a 10 mL flask. Over these solids, 0.5 mL of anhy-
drous CH2Cl2 and 0.5 mL of freshly distilled pyridine were added
using a syringe. After stirring the resulting yellow solution at
25 °C for 20 h, MeOH (0.3 mL) and EtOAc (5 mL) were next added
and the resulting mixture was washed several times with aqueous
saturated CuSO4 solution. The organic phase was dried over MgSO4,
filtered and the solvent removed to give a residue that was purified
by flash chromatography (Hexane/EtOAc 8:2) to afford N-Boc jas-
pines 4a–4c.
5.4.7. Deprotection of N-Boc jaspines 3a–3d
A solution of 0.08 mmol of the corresponding N-Boc jaspine in
1.8 mL of CH2Cl2/TFA (5:1) was stirred for 1 h at 25 °C. The solvent
was next removed under a stream of N2 and the resulting oil was
taken up in aqueous NaHCO3 and extracted with CH2Cl2. The or-
ganic phase was dried over MgSO4, filtered and the solvent was
evaporated under vacuum. The resulting white solid was purified
by flash chromatography (CH2Cl2/CH3OH/NH4OH 96:4:1) to afford
the final compound.
5.4.8. (2R,3S,4S)-4-Amino-2-tetradecyltetrahydrofuran-3-ol (2a)
Obtained in 82% yield (19 mg, 0.06 mmol) as a white solid from
5.4.3. (2R,3S,4S)-4-(tert-Butoxycarbonyl)amino-3-hydroxy-2-
tetradecyltetrahydrofuran (4a)
31 mg (0.08 mmol) of 3a; mp 101–102 °C; ½a D25
ꢂ
+14.8 (c 0.97,
CH3OH); IR (film): 3334, 3281, 3135, 2914, 2853, 1596, 1470,
1367, 1310. 1H NMR (CDCl3, 500 MHz)5: 4.12 (dd, J = 9, 6.5 Hz,
1H, H1a), 3.60 (m, 2H, H3, H4), 3.47 (m, 1H, H2), 3.40 (dd, J = 9,
7 Hz, 1H, H1b), 1.58 (m, 1H), 1.53 (m, 1H), 1.43 (m, 1H), 1.39 (m,
1H), 1.21–1.32 (m, 22H), 0.88 (t, J = 7 Hz, 3H). 13C NMR (CDCl3,
100 MHz): 85.4(C4), 74.9(C3), 73.3 (C1), 52.7 (C2), 33.9, 32.1,
29.8, 29.7, 29.7, 29.5, 26.0, 22.8, 14.3. ESI-MS m/z 300.2 [M+H]+
298.2 [MꢀH]ꢀ; HRMS: calculated for C18H38NO2 [M+H]+:
300.2903; found: 300.2921. HPLC >95% H2O/CH3CN 10:90 (0.1%
TFA) rt = 5.81 min.
Isolated yield: 70%; mp 80–81 °C; ½a D25
ꢂ
+5.0 (c 0.97, CHCl3); IR
(film): 3414, 3370, 2927, 2850, 1691, 1523. 1H NMR (CDCl3,
500 MHz): 5.02 (d, J = 6 Hz, 1H, NH), 4.13 (m, 2H, H2, H1a), 3.92
(m, 1H, H3), 3.70 (ddd, 7.0, 5.9, 4.3 Hz, 1H, H4), 3.50 (t, J = 10 Hz,
1H, H1b), 2.39 (br s, 1H), 1.53 (m, 1H), 1.53 (ddd, J = 8.8, 6.3,
4.3 Hz, 1H), 1.45 (s, 9H), 1.24–1.35 (m, 24H), 0.87 (t, J = 7 Hz,
3H). 13C NMR (CDCl3, 125 MHz): 156.1, 85.3, 80.1, 75.0, 70.3,
53.0, 33.7, 32.1, 29.8, 29.8, 29.8, 29.8, 29.7, 29.7, 29.5, 28.5, 25.9,
22.8, 14.3. ESI-MS m/z, 422.3 [M+Na].
5.4.4. (2S,3R,4S)-4-(tert-Butoxycarbonyl)amino-3-hydroxy-2-
5.4.9. (2S,3R,4S)-4-Amino-2-tetradecyltetrahydrofuran-3-ol (2b)
tetradecyltetrahydrofuran (4b)
Obtained in 82% yield (24 mg, 0.08 mmol) as a white solid from
Isolated yield: 10%; mp 94–96 °C; ½a D25
ꢂ
ꢀ31.7 (c 1.09, CHCl3); IR
39 mg (0.10 mmol) of 3b; mp 77–78 °C; ½a D25
ꢀ3.8 (c 0.71, CHCl3);
ꢂ
(film): 3346, 2915, 2849, 1689, 1550, 1523, 1468, 1390, 1357, 1175.
1H NMR (CDCl3, 500 MHz): 4.83 (d, J = 5.5 Hz, 1H, NH), 4.04 (dd,
J = 9.5, 6.5 Hz, 1H, H1a), 3.91 (m, 1H, H2), 3.76 (dd, J = 5.5, 3.5Hz,
1H, H3), 3.65 (dd, J = 9.5, 3.5 Hz, 1H, H1b), 3.63 (m, 1H, H4), 1.65
(m, 1H), 1.58 (m, 1H), 1.44 (s, 9H), 1.38 (m, 1H), 1.20–1.32 (m,
23H), 0.87 (t, J = 7 Hz, 3H). 13C NMR (CDCl3, 100 MHz): 156.7,
85.1, 82.8, 80.4, 77.4, 70.5, 60.4, 33.8, 32.1, 29.8, 29.8, 29.8, 29.8,
29.7, 29.7, 29.5, 28.5, 26.1, 22.8, 14.3. ESI-MS m/z 422.2 [M+Na].
IR (film): 3359, 3278, 3060, 2924, 2873, 2850, 1586, 1471, 1360,
1243. 1H NMR (CDCl3, 500 MHz): 4.00 (dd, J = 9.5, 6 Hz, 1H, H1a),
3.55–3.61 (m, 3H, H1b, H3, H4), 3.30 (ddd, J = 6, 4.5, 4 Hz, 1H,
H2), 2.16 (br s, 3H), 1.56–1.68 (m, 2H), 1.46 (m, 1H), 1.21–1.40
(m, 23H), 0.87 (t, J = 6.8 Hz, 3H). 13C NMR (CDCl3 + 1 drop CD3OD,
100 MHz): 84.7(C4), 82.5(C3), 72.8(C1), 59.9(C2), 33.9, 32.0, 29.8,
29.8, 29.8, 29.7, 29.7, 29.5, 26.1, 22.8, 14.2. ESI-MS m/z 300.2
[M+H]+, 322.1 [M+Na]; HRMS: calculated for C18H38NO2 [M+H]+:
300.2903; found: 300.2899. HPLC >95% H2O/CH3CN 10:90 (0.1%
TFA) rt = 5.00 min.
5.4.5. (2R,3R,4S)-4-(tert-Butoxycarbonyl)amino-3-hydroxy-2-
tetradecyltetrahydrofuran (4c)
Isolated yield: 50%; mp 106–108 °C; ½a D25
ꢂ
ꢀ22.3 (c 0.82, CHCl3);
5.4.10. (2R,3R,4S)-4-Amino-2-tetradecyltetrahydrofuran-3-ol
(2c)
IR (film): 3385, 3354, 2918, 2847, 1682, 1506, 1465, 1404, 1366,
1343, 1252, 1163. 1H NMR (CDCl3 + 1 drop CD3OD, 500 MHz):
5.11 (d, J = 6 Hz, 1H, NH), 4.04 (dd, J = 9.5, 6 Hz, 1H, H1a), 3.93 (d,
J = 3 Hz, 1H, H3), 3.90 (m, 1H, H2), 3.76 (dt, J = 6.8, 3.8 Hz, 1H,
H4), 3.43 (dd, J = 9.5, 3 Hz, 1H, H1b), 1.63 (m, 2H), 1.44 (s, 9H),
1.38 (m, 1H), 1.20–1.32 (m, 23H), 0.87(t, J = 7 Hz, 3H). 13C NMR
(CDCl3 + 1 drop CD3OD, 100 MHz): 156.1, 81.5, 80.4, 76.7, 70.3,
59.9, 59.8, 32.0, 29.9, 29.8, 29.7, 29.7, 29.7, 29.4, 28.5, 28.4, 26.5,
22.8, 14.2. ESI-MS m/z 422.2 [M+Na], 821.6 [2M+Na].
Obtained in 78% yield (30 mg, 0.10 mmol) as a white solid from
52 mg (0.13 mmol) of 3c; mp 87–88 °C; ½a D25
ꢀ2.5 (c 0.71, CHCl3);
ꢂ
IR (film): 3370, 3303, 2960, 2913, 2853, 1607, 1475, 1293, 1070. 1H
NMR (CDCl3, 500 MHz): 4.20 (dd, J = 9, 6 Hz, 1H, H1a), 3.88 (ddd,
J = 7.5, 6.3, 3.5 Hz, 1H, H4), 3.81 (dd, J = 3, 1.5 Hz, 1H, H3), 3.44
(m, 1H, H2), 3.39 (dd, J = 9, 3.5 Hz, 1H, H1b), 1.60 (m, 5H), 1.43
(m, 1H), 1.20–1.38 (m, 23H), 0.87 (t, J = 7 Hz, 3H). 13C NMR (CDCl3,
100 MHz): 80.9 (C4), 79.6 (C3), 73.4 (C1), 59.9 (C2), 32.0, 29.9, 29.8,
29.7, 29.5, 28.6, 26.5, 22.8, 14.3. ESI-MS m/z 300.2 [M+H]+, 322.1
[M+Na]; HRMS: calculated for C18H38NO2 [M+H]+: 300.2903;
found: 300.2905. HPLC >95% H2O/CH3CN 10:90 (0.1% TFA)
rt = 5.02 min.
5.4.6. (2S,3S,4S)-4-(tert-Butoxycarbonyl)amino-3-hydroxy-2-
tetradecyltetrahydrofuran (4d)
A cooled (0 °C) solution of tosylate 4d (0.40 mmol) in 5 mL of
anhydrous MeOH was treated with 278 mg (2 mmol) of K2CO3
and stirred for 20 h at 25 °C. The solvent was next removed and
the residual white solid was purified by flash chromatography
(Hexane/EtOAc 8:2) to afford 3d in 50% yield. Mp 109–111 °C;
5.4.11. (2S,3S,4S)-4-Amino-2-tetradecyltetrahydrofuran-3-ol (2d)
(jaspine B)
Obtained in 74% yield (25 mg, 0.08 mmol) as a white solid from
lit.17 mp 110–111 °C; ½a 2D5
ꢂ
+6.7 (c 0.89, CHCl3); Lit17
½
a 2D5
ꢂ
+6.7 (c
45 mg (0.11 mmol) of 3d; mp 90–91 °C; ½a D25
ꢂ
+8.7 (c 1.10, CHCl3),
0.89, CHCl3); IR (film): 3365, 2955, 2917, 2849, 1687, 1526, 1470,
1364, 1328, 1241, 1173, 1135. 1H NMR (CDCl3, 500 MHz): 5.07
(br s, 1H, NH), 4.32 (m, 1H, H2), 4.06 (m, 2H, H1a, H3), 3.70 (td,
J = 6.9, 2.8 Hz, 1H, H4), 3.57 (t, J = 8.2 Hz, 1H, H1b), 1.62 (m, 1H),
lit.17
½
a 2D5
+9.5 (c 1.5, CHCl3); IR (film): 3340, 2919, 2854, 1585,
ꢂ
1471, 1365, 1240. 1H NMR4 (CDCl3, 500 MHz)5: 3.92 (dd, J = 8.5,
7.5 Hz, 1H, H1a), 3.86 (dd, J = 4, 3.5 Hz, 1H, H3), 3.73 (ddd, J = 7,
6.7, 3.5 Hz, 1H, H4), 3.65 (m, 1H, H2), 3.50 (dd, J = 8.5, 7 Hz, 1H,

D. Canals et al. / Bioorg. Med. Chem. 17 (2009) 235–241
241
H1b), 1.65 (m, 2H), 1.20–1.42 (m, 24H), 0.87 (t, J = 6.7 Hz, 3H). 13C
NMR (CDCl3, 100 MHz): 83.3 (C4), 72. 5(C1), 71.9 (C3), 54.4 (C2),
32.1, 29.9, 29.8, 29.8, 29.7, 29.5, 29.5, 26.4, 22.8, 14.3. ESI-MS m/z
300.3 [M+H]+, 298.2 [MꢀH]ꢀ. HRMS: calculated for C18H38NO2
[M+H]+: 300.2903; found: 300.2931. HPLC >95% H2O/CH3CN
10:90 (0.1% TFA) rt = 5.87 min.
21. Yakura, T.; Sato, S.; Yoshimoto, Y. Chem. Pharm. Bull. 2007, 55, 1284.
22. Since jaspine diastereomers can be also viewed as anhydro phytosphingosine
derivatives, we were also interested in their evaluation as fungal growth
inhibitors by their analogy with phytosphingosine,
a key sphingolipid in
fungal metabolism. (Thevissen, K.; Francois, I. E.; Aerts, A. M. B.; Cammue, P.
Curr. Drug Targets 2005, 6, 923–928.) Preliminary studies carried out on
Saccharomyces cerevisiae indicated that jaspine B (2d) was inactive as growth
inhibitor at 400 lM, whereas its stereoisomers 2a–2c were moderately
inhibitors, with LD50 values around 400 lM (2a), 80 lM (2b), and 102 lM
(2c).
Acknowledgments
23. Imashiro, R.; Sakurai, O.; Yamashita, T.; Horikawa, H. Tetrahedron 1998, 54,
10657.
Financial support from the Ministerio de Educación y Ciencia,
Spain (Project MCYT CTQ2005-00175/BQU), Fondos Feder (EU),
Fundació La Marató de TV3 (Projects UB-040730 and CSIC-
040731) and Generalitat de Catalunya (2005SGR01063) is
acknowledged. D.M. is acknowledged to the Ministerio de Educa-
ción y Ciencia, Spain, for a predoctoral fellowship.
24. This tosylation–cyclization step is strongly dependent on the stereochemistry
of the starting phytosphingosines. Thus, 4a–4c were obtained in 70%, 10%, and
50% yield from 1a to 1c, respectively (see Section 4). The low yield from 1b
(10%) might be related to energy constrains associated to the transition state of
the cyclization process. Mechanistic studies along this line are in currently
underway to account for these differences.
25. Unpublished results from our group.
26. Kraveka, J. M.; Li, L.; Szulc, Z. M.; Bielawski, J.; Ogretmen, B.; Hannun, Y. A.;
Obeid, L. M.; Bielawska, A. J. Biol. Chem. 2007, 282, 16718.
27. Klionsky, D. J.; Cuervo, A. M.; Seglen, P. O. Autophagy 2007, 3, 181.
28. Klionsky, D. J.; Abeliovich, H.; Agostinis, P.; Agrawal, D. K.; Aliev, G.; Askew, D.
S.; Baba, M.; Baehrecke, E. H.; Bahr, B. A.; Ballabio, A., et al Autophagy 2008, 4,
151.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.11.026.
29. Lavieu, G.; Scarlatti, F.; Sala, G.; Levade, T.; Ghidoni, R.; Botti, J.; Codogno, P.
Autophagy 2007, 3, 45.
30. Merrill, A. H., Jr.; Sullards, M. C.; Allegood, J. C.; Kelly, S.; Wang, E. Methods
2005, 36, 207.
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