Asymmetric synthesis and cytotoxic activity of isomeric phytosphingosine derivatives

Article abstract of DOI:10.1039/c1ob06195j

New phytosphingosine analogues have been conceived, synthesised and their cytotoxicity in B16 murine melanoma cells tested. These compounds embed an isomeric substitution pattern resulting from a formal permutation of the C-2 and C-4 substituents along th

Full text of DOI:10.1039/c1ob06195j

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Organic &
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PAPER
Asymmetric synthesis and cytotoxic activity of isomeric phytosphingosine
derivatives†
Arnaud Rives,a Ce´cile Baudoin-Dehoux,a Nathalie Saffon,b Nathalie Andrieu-Abadiec and Yves Ge´nisson*a
Received 18th July 2011, Accepted 8th September 2011
DOI: 10.1039/c1ob06195j
New phytosphingosine analogues have been conceived, synthesised and their cytotoxicity in B16
murine melanoma cells tested. These compounds embed an isomeric substitution pattern resulting from
a formal permutation of the C-2 and C-4 substituents along the aliphatic skeleton of the original
sphingoid base. Five different stereoisomers have been accessed through regio- and stereocontrolled
opening of the oxirane of long chain epoxyamine precursors. The corresponding N-hexyl and
N-octanoyl derivatives have also been prepared. In cell viability experiments all the primary amines
were found to be more active than the natural phytosphingosine with IC50 in the low mM range for the
most potent compounds.
switch between two major players of cell fate regulation, i.e.
the proapoptotic ceramide and the proliferative and angiogenic
Introduction
sphingosine-1-phosphate.6 Close structural analogues of sphin-
gosine of high anti-cancer pharmacological relevance have thus
been developed, such as the naturally occurring spisulosine7 or
the nature-inspired synthetic safingol,8 and enigmol (Fig. 2).9
Sphingosine represents the archetypal structural backbone com-
mon to all sphingolipids. Two main C18 derivatives are typically
encountered in living organisms: D-erythro-sphingosine or D-ribo-
phytosphingosine (Fig. 1).1 Due to the strong contribution of
sphingolipids as signalling molecules in cell growth and differenti-
ation regulation,2 the sphingosine framework has inspired intense
synthetic studies.3
Fig. 1 Structure of the two most widespread sphingoid bases.
D-erythro-Sphingosine, the most common form in mammals,
is ubiquitous in eukaryotic cells. It has been described to induce
cell cycle arrest and apoptosis,4 through modulation of protein
kinases.5 Additionally, this sphingoid base constitutes a metabolic
Fig. 2 Structure of pharmacologically relevant sphingosine analogues.
D-ribo-Phytosphingosine is more frequent in plants and fungi.10
In yeast it has been identified as a signalling molecule involved
in the cell cycle arrest in response to heat stress.11 It is also
encountered in mammals, such as in intestinal12 and skin cells.13
Phytosphingosine has been shown to have a pro-apoptotic effect
on different cancer cell lines, either alone,14 or in combination
with other treatments.15 Related phytoceramides, obtained upon
N-acylation, were described to be more cytotoxic than the
corresponding sphingosine-derived ceramides.16 Other important
biologically active phytosphingosine-related compounds have also
been reported,17 including cyclised analogues.18
aLSPCMIB, UMR 5068, CNRS-Universite´ Paul Sabatier - Toulouse III,
Toulouse, France. E-mail: genisson@chimie.ups-tlse.fr; Fax: +33 (0)5 61
55 60 11; Tel: +33 (0)5 61 55 62 99
bInstitut de Chimie de Toulouse, ICT FR 2599, Universite´ Paul Sabatier -
Toulouse III, Toulouse, France
cCRCT, INSERM UMR-1037, Universite´ Paul Sabatier - Toulouse III,
CHU Rangueil, Toulouse, France
† Electronic supplementary information (ESI) available: copies of the
1H NMR spectra for 2,4, 6–18 and 13C NMR spectra for 6–18. CCDC
reference numbers 836021 and 836022. For ESI and crystallographic data
in CIF or other electronic format see DOI: 10.1039/c1ob06195j
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In the course of our recent work on the preparation of
aza-analogues of the anhydrophytosphingosine jaspine B, we
developed a straightforward access to enantioenriched long-chain
sphingosine-related epoxyamines (Scheme 1).19 We envisioned
that these intermediates could give rise to phytosphingosine-
like aminotriol derivatives upon formal epoxide hydrolysis. In
particular, the location of the amino group would lead to an
unprecedented isomeric substitution pattern resulting from a
formal permutation of the C-2 and C-4 substituents along the
aliphatic skeleton of the original sphingoid base. We will refer to
these entities as “isophytosphingosines” in the rest of the article.
Scheme 2 Preparation of the epoxyamines. Reagents and conditions: a) i)
◦
◦
˚
BnNH2, 4 A MS, Et2O, rt, 18 h, 70%; ii) Et2OBF3, THF, -78 C to -40 C
then C14H29MgCl, -78 ◦C, 2 h, 53%.
Access to 3,4-anti aminotriols
Starting from 3,4-anti epoxyamines, a regio- and stereocontrolled
C-2 epoxide hydrolysis was expected to yield 2,3-syn/3,4-anti or
2,3-anti/3,4-anti from a cis or a trans epoxide respectively. The
cis epoxyamine 2 was first desilylated and the resulting primary
alcohol 5 was treated with diluted H2SO4 in dioxane (Scheme
3). To our satisfaction, these conditions, previously developed for
vinyl derivatives, proved equally efficient in the aliphatic series.22
The expected aminotriol 6 was isolated in 66% yield. Only traces
(ca. 5%) of another isomer were detected in HPLC/MS analysis
of the crude mixture (m/z 408). The same procedure was directly
applied to the O-silylated trans epoxyalcohol 4 to selectively deliver
the expected 2,3-anti/3,4-anti isomer 7 in 65% yield.
Scheme 1 General approach.
We thus report herein the enantioselective preparation of
long chain 4-amino-1,2,3-triols as well as several N-substituted
derivatives thereof. The present study includes the first biological
evaluation of these novel isophytosphingosine derivatives regard-
ing their effect on melanoma cell viability.
Results and discussion
Synthetic plan
Taking advantage of the flexibility of our synthetic route, we
targeted a panel of stereoisomeric forms. Indeed, there is typically a
strong influence of the configuration of the sphingosine analogues
on their bioactivity. For example the ceramide synthase inhibitory
activity of a series of 1-deoxy sphingosine analogues, including
enigmols, was shown to strongly depend on their stereochemical
pattern.20 Our plan was to rely on a stereochemical manifold based
on the following key elements: 1) the configuration of the starting
epoxide; 2) the 3,4-anti epoxyamine relationship and 3) the regio-
and stereocontrolled C-2 or C-3 epoxide opening.21
Scheme 3 Hydrolysis of the epoxyamines toward 3,4-anti aminotriols.
Reagents and conditions: a) TBAF/SiO2, THF, rt, 16 h, 81%. b) 3 M
H2SO4, 1,4-dioxane, reflux, 5 h, 66–65%. c) 12 bar H2, Pd(OH)2, MeOH,
72 h, 77–95%.
Gratifyingly, a crystal sample of the latter proved suitable
for X-ray diffraction analysis. The resulting structure allowed
unambiguous confirmation of the (2S*,3R*,4R*) configuration
(Fig. 3).
A final hydrogenolysis afforded the targeted isophytosphin-
gosines 10 and 11 in high yields (Scheme 3). Ent-10 was prepared
from ent-2 using a reaction sequence identical to that employed
for 10.
Preparation of the starting epoxyamines
The representative starting epoxyamines were obtained via the
anti-selective addition of the tetradecyl Grignard reagent onto an
intermediate epoxyimine (Scheme 2).19
Access to 3,4-syn aminotriols
The cis and trans derivatives 2 and 4 were obtained in 70% (dr
> 95 : 5) and 53% (dr 80 : 20) yields respectively from the starting
epoxyaldehyde. The cis epoxyamine ent-2 was prepared in a similar
fashion from the enantiomeric aldehyde precursor.
The preparation of 3,4-syn aminotriols from 3,4-anti epoxyamines
required an inversion of configuration at C-3. This was planned
through the implementation of our carbonation/intramolecular
cyclisation sequence ensuring neat C-3 epoxide opening.24
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activity has been recently evidenced.26 Depending on the nature
of the N-substituent, additional physicochemical factors such as
the hydrophobicity of double-chained derivatives may also be
involved. In the context of the present study, two representative
transformations were selected, i.e. the N-alkylation and the N-
acylation. An aliphatic residue of moderate chain length (C6 to
C8) was selected in both cases so as to mimic ceramide while
retaining reasonable hydrophilicity.
Applying a general procedure of reductive amination with n-
hexanal, the four N-hexyl amines 13, 14, ent-13, ent-14 were ob-
tained in reasonable yields from the corresponding isophytosphin-
gosines (Scheme 5). Alternatively, N-acylation was conducted with
p-nitrophenyl octanoate to give the expected isophytoceramides
15–17, ent-15 and ent-16 with good efficiency. For the sake of
comparison, the natural phytoceramide 18 was prepared similarly
from commercially available D-ribo-phytosphingosine.
Fig. 3 Molecular view of the (2S,3R,4R)-aminotriol 7 in the solid state
(thermal ellipsoids at 50% probability); hydrogens are omitted for clarity
except on asymmetric carbons.23
Treatment of the deprotected cis epoxyamine 5 with ammonium
carbonate in THF/H2O led to the efficient and selective formation
of the desired oxazolidinone 8 (Scheme 4).
Scheme 4 The oxazolidinone route towards 3,4-syn aminotriols. Reagents
and conditions: a) (NH4)2CO3, THF/H2O, rt, 8 h, 95%. b) NaOH,
EtOH/H2O, reflux, 6 h, 83%. c) 12 bar H2, Pd(OH)2, MeOH, 72 h, 62%.
The latter was saponified to give the 2,3-syn/3,4-syn aminotriol
9 in 83% yield. Again, a crystal structure of this N-benzyl inter-
mediate was obtained by X-ray diffraction analysis confirming the
(2R*,3R*,4S*) stereochemical assignment (Fig. 4).
Scheme
5
Preparation of N-functionalised (iso)phytosphingosines.
Reagents and conditions: a) n-hexanal, NaBH3CN, HCl conc., MeOH,
rt, 16 h, 50–65%. b) p-nitrophenyl octanoate, THF, rt, 48 h, 60–63%.
Biological evaluation
Fig. 4 Molecular view of the (2R,3R,4S)-aminotriol 9 in the solid state
(thermal ellipsoids at 50% probability); hydrogens are omitted for clarity
except on asymmetric carbons.25
Compounds were evaluated for their capacity to alter murine
melanoma B16 cells viability. Melanoma is indeed considered
as a radiation-, immunotherapy-, and chemotherapy-refractory
neoplasm.
Regarding the amide derivatives, none of the five compounds
15–17, ent-15 and ent-16 tested displayed a significant cytotoxicity
at concentrations up to 50 mM. Surprisingly, the same trend
was observed for the natural D-ribo-phytosphingosine-derived
ceramide 18 (up to 25 mM).
Hydrogenolytic N-benzyl deprotection afforded the expected
isophytosphingosine 12. Ent-12 was similarly obtained starting
from the epoxyamine ent-5.
N-Functionalisation of the aminotriols
In modulating the steric and electronic environment of the nitrogen
atom, N-functionalisation of sphingoid bases deeply impacts their
biological profile. For example, a direct relationship between the
degree of N-methylation of the sphingosine and its signalling
Among the four N-alkyl derivatives evaluated, the 4R com-
pounds ent-13 and ent-14 proved slightly more active (40–50%
cytotoxicity at 10 mM) than the 4S stereoisomers 13 and 14 (30–
35% cytotoxicity at 10 mM).
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The more significant activities were recorded in the unsubsti-
tuted isophytosphingosine series. Apart from the 4S derivative 10
(50% cytotoxicity at 10 mM), all the primary amines showed fair
cytotoxicity (70–79% at 10 mM). The two more potent compounds
ent-10 and 11 were evaluated at different concentrations. The all-R
ent-10 displayed a dose-dependent effect with an IC50 of 3 mM. The
(2S,3R,4R) isomer 11, possessing a stereochemical pattern anal-
ogous to that of the natural D-ribo-phytosphingosine, displayed
a similar behaviour, whereas the natural phytosphingosine itself
gave an IC50 of 15 mM in B16 cells.
the diffractometer. The data were integrated with SAINT, and an
empirical absorption correction with SADABS was applied.27,28
The structures were solved by direct methods, using SHELXS-
9729 and refined using the least–squares method on F2.30 All
non-H atoms were treated anisotropically. 3-(4,5-Dimethylthiazol-
2-yl)-5-diphenyltetrazolium bromide (MTT) was supplied from
Euromedex. D-ribo-Phytosphingosine was purchased from Aventi
Polar Lipids. Murine B16 melanoma cell line was purchased from
American Type Culture Collection.
General procedures
Conclusions
General procedure for the epoxide hydrolysis. A 3 M aqueous
H2SO4 solution (8.00 eq) was added to a solution of product
(1.00 eq) in 1,4-dioxane (0.2 M). The mixture was refluxed for 4 h
and allowed to cool down before neutralisation with 3 M aqueous
NaOH solution (16.5 eq) followed by solid NaHCO3. 1,4-Dioxane
was then evaporated off under reduced pressure and the resulting
aqueous phase extracted with EtOAc. The combined organic
phases were successively washed with water and brine, dried over
Na2SO4, filtered and concentrated under reduced pressure.
The extension of our regio- and stereocontrolled epoxide opening
procedures to long chain cis or trans anti-epoxyamines led to novel
isomeric phytosphingosine analogues. The 3,4-anti series was
accessed via acidic hydrolysis while the use of oxazolidine interme-
diates allowed preparation of the 3,4-syn analogues. Five different
stereoisomers were thus prepared and N-functionalisation was
also explored through the preparation of four N-hexyl and five
N-octanoyl derivatives. Cell viability experiments showed that
the amide series was inactive and that the secondary amines
displayed only modest cytotoxicity. On the other hand, all the
primary amines were revealed to be more active than the natural
phytosphingosine with the most potent derivatives being the all-
R and the (2S,3R,4R) isomers ent-10 and 11. The present work
opens up prospects for further studies aiming at synthesizing other
stereoisomers and elucidating the mode of action of these novel
phytosphingosine-type derivatives.
General procedure for the hydrogenolysis. 10% Pd(OH)2/C
(20–30% w/w) and 12 N HCl aqueous solution (1–2 drops)
were successively added to a solution of N-benzylamine (1.00 eq)
in MeOH (0.1 M). The flask was purged with N2 and then
loaded with H2 (10–12 bars). The mixture was stirred at room
temperature until disappearance of the starting material (24–90
R
h). The catalyst was then removed by filtration through Celiteꢀ
and the filtrate evaporated to dryness. The residue was taken
up in 2 : 1 MeOH/water (25 mL mmol-1) and Dowex 50WX8-
200 ion-exchange resin (12.0 g mmol-1) was added. After being
stirred for 1 h, the resin was successively filtered and washed with
water and MeOH. A 3 M ammonium hydroxide solution was then
added (50 mL mmol-1) and the suspension was stirred for 1 h
before being filtered and rinsed with a 3 M ammonium hydroxide
solution (500 mL mmol-1). The resulting solution was evaporated
to dryness under reduced pressure.
Experimental section
General considerations
The following solvents and reagents were dried prior to use:
CH2Cl2, MeOH, DMF (from calcium hydride), 1,2-dimethoxy-
ethane, Et2O, petroleum ether, THF, toluene (freshly distilled
from sodium/benzophenone), Et3N (from calcium hydride, stored
over KOH pellets). Analytical thin layer chromatography (TLC)
was performed using Merck silica gel 60F254 precoated plates.
Chromatograms were observed under UV light and/or were
visualised by heating plates that were dipped in 10% phos-
phomolybdic acid in EtOH or Dragendorff reagent. Column
chromatography was carried out with SDS 35–70 mm flash
silica gel. NMR spectroscopic data were obtained with Bruker
Avance 300. Chemical shifts are quoted in parts per million
(ppm) relative to residual solvent peak. J values are given in Hz.
Infrared (IR) spectra were recorded on a Perkin–Elmer FT-IR
1725X spectrometer. Mass spectrometry (MS) data were obtained
on a ThermoQuest TSQ 7000 spectrometer. High-resolution
mass spectra (HRMS) were performed on a ThermoFinnigan
MAT 95 XL spectrometer. LC–MS analyses and preparative
HPLC purification were performed using a Waters Autopurif
apparatus. Optical rotations were measured on a Perkin–Elmer
model 241 spectrometer. Crystallographic data were collected
at low temperature (193 K) on a Bruker SMART Apex II
diffractometer with graphite-monochromated Mo-Ka radiation
General procedure for the reductive amination. To aminotriol
hydrochloride (prepared by treating the free base with a methano-
lic solution of HCl or obtained as the crude product of hydrogenol-
ysis) (1.00 eq) in MeOH (0.07 M) at room temperature and under
nitrogen atmosphere was added NaBH3CN (1.60 eq) and hexanal
(1.00 eq). The mixture was stirred for 16 h after which it was
diluted with water. The mixture was extracted three times with
EtOAc and the combined extracts were washed with brine, dried
over Na2SO4 and the solvent was evaporated in vacuo.
General procedure for the N-acylation. To a solution of
aminotriol (1.00 eq) in anhydrous THF (0.03 M) at room tem-
perature and under nitrogen atmosphere was added 4-nitrophenyl
caprylate (2.00 eq). The mixture was stirred for 2 days after which
the solvent was removed in vacuo.
Specific procedures and characterisation data
((2R,3S)-3-((S)-1-(Benzylamino)pentadecyl)oxiran-2-yl)metha-
nol (5). TBAF on silica gel (6.97 g at ca. 1.25 mole of fluoride/g,
ca. 8.71 mmol, ca. 2 eq) was added to a solution of 2 (2.69 g,
4.38 mmol) in anhydrous THF (44 mL). The reaction mixture
˚
(l = 0.71073 A) by using phi- and omega-scans. Single crystals
were mounted in inert oil and transferred to the cold gas stream of
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was vigorously stirred until TLC analysis showed no remaining
starting material (ca. 16h) and then filtrated. Silica gel was
rinsed several times with EtOAc (3 ¥ 50 mL) and the combined
filtrates concentrated in vacuo. The resulting crude product was
purified by flash column chromatography on silica gel eluting with
CH2Cl2/MeOH (98 : 2) to afford primary alcohol 5 (1.34 g, 81%)
(4R,5R)-3-Benzyl-5-((R)-1,2-dihydroxyethyl)-4-tetradecyloxa-
zolidin-2-one (8). To a solution of epoxyamine 5 (200 mg, 0.53
mmol) in a 4 : 1 THF/water mixture (2 mL) was added (NH4)2CO3
(407 mg, 4.24 mmol, 8.00 eq) and the heterogeneous mixture
was vigorously stirred at room temperature until TLC analysis
showed disappearance of the starting material. The THF was then
evaporated off under reduced pressure and the resulting aqueous
phase extracted with Et2O. The combined organic phases were
successively washed with water and brine, dried over Na2SO4,
filtered and concentrated under reduced pressure. The crude
product was purified by flash column chromatography on silica
gel (CH2Cl2/MeOH 95 : 5) to give 8 (205 mg, 95%). Rf = 0.28
(CH2Cl2/MeOH 95 : 5). [a]D25 +58.1 (c 1.1, CHCl3). IR (neat) 3365
(O–H), 1728 (C O) cm-1. 1H NMR (300 MHz, CDCl3) d 0.86 (t,
3J = 6.9 Hz, 3H), 1.05–1.29 (m, 24H), 1.39–1.55 (m, 2H), 3.55–3.75
25
as a colourless oil. Rf = 0.26 (CH2Cl2/MeOH 95 : 5). [a]D -13.4
(c 0.9, CHCl3). IR (neat) 3435 (O–H), 1042 (C–O) cm-1.1H NMR
(300 MHz, CDCl3) d 0.85 (t, 3J = 6.6 Hz, 3H), 1.10–1.48 (m, 24H),
1.52–1.84 (m, 2H), 2.37–2.44 (m, 1H), 2.91 (dd, 3J = 8.6 Hz, 3J =
2
3
4.1 Hz, 1H), 3.18–3.24 (m, 1H), 3.52 (dd, Jgem = 11.0 Hz, J =
6.9 Hz, 1H, H1) 3.69–3.73 (AB system, 2Jgem = 12.4 Hz, da - db =
17.7 Hz, 2H), 3.86 (dd, 2Jgem = 11.0 Hz, 3J = 5.2 Hz, 1H), 7.24–7.40
(m, 5H). 13C NMR (75 MHz, CDCl3) d 14.1, 22.6, 25.1, 29.3–29.7
(9C), 31.9, 32.5, 50.5, 55.4, 59.5, 60.9, 127.4, 128.3, 128.6, 139.0.
MS (ES): m/z = 390 (100%) [M + H+]. HRMS (ESI+): C25H44NO2
calc. 390.3405, found 390.3372.
2
(m, 5H) 4.09 (d, J = 15.5 Hz, 1H), 4.19–4.21 (m, 1H), 4.67 (d,
2J = 15.5 Hz), 7.20–7.31 (m, 5H). 13C NMR (75 MHz, CDCl3) d
14.0, 22.6, 23.4, 29.3–29.6 (9C), 31.4, 31.9, 46.1, 56.1, 63.0, 72.5,
78.7, 127.7, 127.8, 128.6, 139.5, 158.3. MS (ES): m/z 456 (100%)
[M + Na+]. HRMS (ESI+): C26H43NO4Na calc. 456.3090, found
456.3083.
((2S,3R)-3-((R)-1-(Benzylamino)pentadecyl)oxiran-2-yl)metha-
nol (ent-5). Compound ent-5 was prepared in the same way as
its enantiomer. It displayed identical analytical data expect for its
25
optical rotation. [a]D +12.6 (c 1.1, CHCl3).
(4R,5S)-3-Benzyl-5-((S)-1,2-dihydroxyethyl)-4-tetradecyloxa-
zolidin-2-one (ent-8). Compound ent-8 was prepared in the same
way as its enantiomer. It displayed identical analytical data expect
(2S,3S,4S)-4-(Benzylamino)octadecane-1,2,3-triol (6). Epo-
xyamine 5 (300 mg, 0.79 mmol) was reacted according to the
general procedure for epoxide hydrolysis. The crude product
was purified by flash column chromatography on silica gel
(EtOAc/Ether/MeOH 85 : 10 : 5, 1.2% NH4OH) to give 6 (210 mg,
66%). Rf = 0.10 (EtOAc/Ether/MeOH 85 : 10 : 5, 1.2% NH4OH).
25
for its optical rotation. [a]D -57.6 (c 1.4, CHCl3).
(2R,3R,4S)-4-(Benzylamino)octadecane-1,2,3-triol
(9).
NaOH (92.0 mg, 2.30 mmol, 10.0 eq) was added to a solution
[a]D -6.3 (c 0.7, CHCl3). IR (neat) 3400 (O–H), 1056 (C–O) cm-1.
of oxazolidinone 8 (98.0 mg, 0.23 mmol) in a 8 : 2 EtOH/H2O
25
◦
1H NMR (300 MHz, CDCl3) d 0.85 (t, 3J = 6.7 Hz, 3H), 1.05–1.29
(m, 24H), 1.35–1.54 (m, 2H), 2.86–2.92 (m, 1H), 3.74–3.89 (m,
6H) 7.28–7.33 (m, 5H). 13C NMR (75 MHz, CDCl3) d 14.1, 22.6,
26.2, 29.3, 29.4, 29.5, 29.6–29.7 (6C), 30.7, 31.9, 52.9, 60.8, 65.5,
70.3, 73.6, 127.2, 128.5, 128.6, 139.5. MS (ES): m/z 408 (100) [M
+ H+]. HRMS (ESI+): C25H46NO3 calc. 408.3478, found 408.3487
mixture (4.6 mL). The reaction mixture was heated at 85 C for
6 h before being cooled to room temperature. The mixture was
extracted three times with EtOAc and the combined extracts were
concentrated in vacuo. The crude product was purified by flash
column chromatography on silica gel (CH2Cl2/MeOH 95 : 5) to
25
give 9 (76 mg, 82%). Rf = 0.17 (CH2Cl2/MeOH 95 : 5). [a]D -51.3
(c 0.8; CHCl3). IR (neat) 3420 (O–H), 2892 (C–H) cm-1. 1H NMR
(2R,3R,4R)-4-(Benzylamino)octadecane-1,2,3-triol
(ent-6).
3
(300 MHz, CD3OD + traces of CDCl3) d 0.87 (t, J = 6.9 Hz,
Compound ent-6 was prepared in the same way as its enantiomer.
3H), 1.19–1.45 (m, 24H), 1.59–1.74 (m, 2H), 2.83–2.89 (m, 1H),
It displayed identical analytical data expect for its optical rotation.
2
3.59–3.62 (m, 2H), 3.64–3.72 (m, 2H), 3.82 (d, J = 12.7 Hz),
25
[a]D +6.1 (c 1.1, CHCl3).
2
4.03 (d, J = 12.7 Hz, 1H), 7.24–7.38 (m, 5H). 13C NMR (75
(2S,3R,4R)-4-(Benzylamino)octadecane-1,2,3-triol (7). trans-
Epoxyamine 4 (507 mg, 0.81 mmol) was reacted according to
the general procedure for epoxide hydrolysis. The crude product
was purified by flash column chromatography on silica gel
(CH2Cl2/MeOH 95 : 5) to give 7 (180 mg, 65%). Rf = 0.18
MHz, CD3OD + traces of CDCl3) d 14.4, 23.5, 27.0, 30.1, 30.2,
30.4–30.6 (7C), 32.8, 51.2, 61.6, 63.9, 69.6, 75.3, 128.8, 129.5,
129.6, 138.0. MS (ES): m/z 408 (100%) [M + H+]. HRMS (ESI+):
C25H46NO3 calc. 408.3478, found 408.3439. Selected crystal data
for 9: C25H45NO3, M = 407.62, Monoclinic, space group P21, a◦=
25
˚
˚
˚
(CH2Cl2/MeOH 95 : 5). [a]D -14.2 (c 1.0, CHCl3). IR (neat) 3376
10.2997(5) A, b = 4.9870(3) A, c = 24.6551(13) A, b = 92.668(4) ,
1
3
3
(O–H), 1051 (C–O) cm-1. H NMR (300 MHz, MeOD + traces
V = 1265.03(12) A , Z = 2, crystal size 0.60 ¥ 0.40 ¥ 0.10 mm ,
17 243 reflections collected (5095 independent, Rint = 0.0388),
321 parameters, R1 [I > 2s(I)] = 0.0545, wR2 [all data] = 0.1557,
˚
of CDCl3) d 0.85 (t, 3J = 6.7 Hz, 3H), 1.05–1.29 (m, 24H), 1.35–
1.54 (m, 2H), 2.79–2.82 (m, 1H), 3.53–3.84 (m, 6H) 7.15–7.34
(m, 5H). 13C NMR (75 MHz, MeOD + traces of CDCl3) d 14.4,
23.1, 25.7, 29.7, 29.9, 30.2–30.5 (8C), 32.5, 51.8, 61.0, 63.7, 71.7,
74.5, 127.9, 128.9, 129.1, 139.7. MS (ES): m/z 408 (100%) [M +
H+]. HRMS (ESI+): C25H46NO3 calc. 408.3478, found 408.3471.
Selected crystal data for 7: C25H45NO3, M = 407.62, Orthorhombic,
-3
˚
largest diff. peak and hole: 0.200 and -0.170 e A .
(2S,3S,4R)-4-(Benzylamino)octadecane-1,2,3-triol
(ent-9).
Compound ent-9 was prepared in the same way as its enantiomer.
It displayed identical analytical data expect for its optical rotation.
25
[a]D +50.2 (c 0.8, CHCl3).
˚
˚
space group P212121, a = 4.9152(2) A, b = 9.6665(4) A, c =
3
˚
˚
50.7258(18) A, V = 2410.12(16) A , Z = 4, crystal size 0.42 ¥
0.10 ¥ 0.04 mm3, 25 744 reflections collected (4399 independent,
Rint = 0.1026), 269 parameters, R1 [I > 2s(I)] = 0.0509, wR2 [all
(2S,3S,4S)-4-Aminooctadecane-1,2,3-triol
(10). N-Benzyl
amine 6 (34.0 mg, 0.09 mmol) was reacted according to the
general procedure for hydrogenolysis. The crude product
was purified by flash column chromatography on silica gel
-3
˚
data] = 0.1049, largest diff. peak and hole: 0.164 and -0.153 e A .
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(MeOH/EtOH/NH4OH/CH2Cl2 3 : 12 : 6 : 79) to give 10 (24 mg,
95%). Rf = 0.22 (MeOH/EtOH/NH4OH/CH2Cl2 12 : 12 : 6 : 70).
[a]D25 +2.3 (c 1.1, MeOH). IR (neat) 3426 (O–H), 2917 (C–H) cm-1.
MS (ES): m/z = 402 (100%) [M + H+]. HRMS (ESI+): C24H52NO4
calc. 402.3947, found 402.3987.
(2R,3R,4R)-4-(Hexylamino)octadecane-1,2,3-triol
(ent-13).
3
1H NMR (300 MHz, CD3OD + traces of CDCl3) d 0.88 (t, J =
Compound ent-13 was prepared in the same way as its
6.3 Hz, 3H), 1.31–1.42 (m, 24H), 1.43–1.73 (m, 2H), 2.82–2.92
(m, 1H), 3.38 (dd, 3J = 6.3 Hz, 3J = 2.4 Hz 1H), 3.54–3.64 (m, 2H),
3.75–3.79 (m, 1H). 13C NMR (75 MHz, CD3OD) d 14.5, 23.8,
27.2, 30.5, 30.8–30.9 (7C), 31.1, 33.1, 34.1, 54.9, 64.9, 72.8, 74.5.
MS (ES): m/z 318 (100%) [M + H+]. HRMS (ESI+): C18H40NO3
calc. 318.3008, found 318.3042.
enantiomer. It displayed identical analytical data expect for its
25
optical rotation. [a]D +12.3 (c 0.8, MeOH).
(2R,3R,4S)-4-(Hexylamino)octadecane-1,2,3-triol (14). Ami-
notriol hydrochloride 12 (35.6 mg, 0.10 mmol) was reacted accord-
ing to the general procedure for procedure for reductive amination.
The crude material was purified by column chromatography on
silica gel eluted with EtOAc/MeOH 98 : 2 to give 14 (27.9 mg,
65%) as a white amorphous solid. Rf = 0.3 (EtOAc/MeOH 95 : 5).
(2R,3R,4R)-4-Aminooctadecane-1,2,3-triol
(ent-10). Com-
pound ent-10 was prepared in the same way as its enantiomer. It
displayed identical analytical data expect for its optical rotation.
25
[a]D -31.8 (c 1.2, MeOH). IR (neat) 3408 (O–H), 2924 (C–H)
25
[a]D -2.1 (c 1.0, MeOH).
1
cm-1. H NMR (300 MHz, CD3OD) d 0.84–1.04 (m, 6H), 1.26–
1.70 (m, 36H) 2.44–2.62 (m, 1H), 2.66–2.76 (m, 1H), 2.76–2.94 (m,
1H), 3.58–3.68 (m, 3H), 3.70–3.82 (m, 1H). 13C NMR (75 MHz,
CD3OD) d 14.4, 14.5, 23.7, 23.8, 27.4, 28.1, 30.5, 30.7, 30.8 (2
peaks), 31.0, 31.1, 31.3, 33.0, 33.1, 48.0, 62.0, 64.3, 70.7, 75.6. MS
(ES): m/z 402 (100%) [M + H+]. HRMS (ESI+): C24H52NO4 calc.
402.3947, found 402.3965.
(2S,3R,4R)-4-Aminooctadecane-1,2,3-triol
(11). N-Benzyl
amine 7 (76.5 mg, 0.09 mmol) was reacted according to the
general procedure for hydrogenolysis. The crude product
was purified by flash column chromatography on silica gel
(MeOH/EtOH/NH4OH/CH2Cl2 3 : 12 : 6 : 79) to give 11
(24.0 mg, 77%). Rf = 0.22 (MeOH/EtOH/NH4OH/CH2Cl2
25
12 : 12 : 6 : 70). [a]D +2.3 (c 1.1, MeOH). IR (neat) 3426 (O–H),
(2S,3S,4R)-4-(Hexylamino)octadecane-1,2,3-triol
(ent-14).
1
3
2917 (C–H) cm-1. H NMR (300 MHz, CD3OD) d 0.88 (t, J =
6.3 Hz, 3H), 1.31–1.42 (m, 24H), 1.43–1.73 (m, 2H), 2.88–2.94 (m,
1H), 3.42–3.46 (m, 1H), 3.54–3.63 (m, 2H), 3.68–3.76 (m, 1H).
13C NMR (75 MHz, CD3OD) d 14.4, 23.2, 26.6, 30.0–30.4 (9C),
33.1, 34.1, 54.5, 63.8, 73.8, 74.9. MS (ES): m/z 318 (100%) [M +
H+]. HRMS (ESI+): C18H40NO3 calc. 318.3008, found 318.3024.
Compound ent-14 was prepared in the same way as its
enantiomer. It displayed identical analytical data expect for its
25
optical rotation. [a]D +29.4 (c 1.2, MeOH)
N-((2S,3S,4S)-1,2,3-Trihydroxyoctadecan-4-yl)octanamide (15).
Aminotriol 10 (31.0 mg, 0.10 mmol) was reacted according
to the general procedure for N-acylation. The crude material
was purified by column chromatography on silica gel eluted
with EtOAc/MeOH 98 : 2 to give 15 (26.2 mg, 63%). Rf = 0.3
(2R,3R,4S)-4-Aminooctadecane-1,2,3-triol
(12). N-Benzyl
amine 9 (31.0 mg, 0.08 mmol) was reacted according to the
general procedure for hydrogenolysis. The crude product
was purified by flash column chromatography on silica gel
(MeOH/EtOH/NH4OH/CH2Cl2 3 : 12 : 6 : 79) to give 12 (15 mg,
62%). Rf = 0.27 (MeOH/EtOH/NH4OH/CH2Cl2 12 : 12 : 6 : 70).
25
(EtOAc/MeOH 95/5). [a]D +1.9 (c 1.0, MeOH). IR (neat) 3405
(O–H), 2851 (C–H), 1631 (C O) cm-1. 1H NMR (300 MHz,
CD3OD) d 0.88–0.98 (m, 6H), 1.24–1.54 (m, 34H) 1.58–1.74 (m,
3
2H), 2.26 (t, J = 7.2 Hz, 2H), 3.34–3.36 (m, 1H), 3.60–3.65 (m,
25
3H), 3.86–3.93 (m, 1H). 13C NMR (75 MHz, CD3OD) d 14.5, 23.8
(2 peaks), 27.3 (2 peaks), 30.3, 30.4, 30.5, 30.6, 30.8 (3 peaks), 31.7,
33.0, 33.1, 37.2, 52.6, 64.4, 72.2, 74.2, 177.2. MS (ES): m/z 466
(100%) [M + Na+]. HRMS (ESI+): C26H53NO4Na calc. 466.3872,
found 466.3896.
[a]D +4.2 (c 1.1, MeOH). IR (neat) 3425 (O–H), 2905 (C–H)
1
3
cm-1. H NMR (300 MHz, CD3OD) d 0.90 (t, J = 6.4 Hz, 3H),
1.24–1.47 (m, 24H), 1.48–1.65 (m, 2H), 2.80–2.90 (m, 1H), 3.45
3
3
(dd, J = 4.4 Hz, J = 2.6 Hz, 1H), 3.57–3.63 (m, 2H), 3.65–3.72
(m, 1H). 13C NMR (75 MHz, CD3OD) d 14.5, 23.8, 27.2, 30.5,
30.8–30.9 (7C), 31.1, 33.1, 35.4, 54.6, 64.4, 73.5, 74.5. MS (ES):
m/z 318 (100%) [M + H+]. HRMS (ESI+): C18H40NO3 calc.
318.3008, found 318.2997.
N-((2R,3R,4R)-1,2,3-Trihydroxyoctadecan-4-yl)octanamide (ent-
15). Compound ent-15 was prepared in the same way as its
enantiomer. It displayed identical analytical data expect for its
25
(2S,3S,4R)-4-Aminooctadecane-1,2,3-triol
pound ent-12 was prepared in the same way as its enantiomer. It
(ent-12). Com-
optical rotation. [a]D -1.6 (c 1.2, MeOH).
N-((2R,3R,4S)-1,2,3-Trihydroxyoctadecan-4-yl)octanamide (16).
Aminotriol 12 (33.0 mg, 0.10 mmol) was reacted according to
the general procedure for N-acylation. The crude material was
purified by column chromatography on silica gel eluted with
EtOAc/MeOH 98 : 2 to give 16 (28.4 mg, 62%) as a white
displayed identical analytical data expect for its optical rotation.
25
[a]D -4.1 (c 1.1, MeOH).
(2S,3S,4S)-4-(Hexylamino)octadecane-1,2,3-triol (13). Ami-
notriol hydrochloride 10 (51.0 mg, 0.14 mmol) was reacted
according to the general procedure for reductive amination. The
crude material was purified by column chromatography on silica
gel eluted with EtOAc/MeOH 98 : 2 to give 13 (28.1 mg, 50%).
25
amorphous solid. Rf = 0.3 (EtOAc/MeOH 95 : 5). [a]D +7.9 (c
1.1, MeOH). IR (neat) 3405 (O–H), 2919 (C–H), 1631 (C O)
1
cm-1. H NMR (300 MHz, CD3OD) d 0.84–0.94 (m, 6H), 1.24–
25
Rf = 0.32 (EtOAc/MeOH 95/5). [a]D -11.0 (c 1.5, MeOH). IR
1.42 (m, 34H), 1.50–1.70 (m, 2H), 2.21 (t, 3J = 7.2 Hz, 2H), 3.46–
3.58 (m, 2H), 3.59–3.68 (m, 2H), 3.94–4.03 (m, 1H). 13C NMR (75
MHz, CD3OD) d 14.5, 23.8 (2 peaks), 27.3 (2 peaks), 30.3, 30.4,
30.5, 30.6, 30.8 (3 peaks), 33.0, 33.1, 33.2, 37.3, 52.1, 64.4, 73.7,
74.0, 176.3. MS (ES): m/z 466 (100%) [M + Na+]. HRMS (ESI+):
C26H53NO4Na calc. 466.3872, found 466.3896.
(neat) 3407 (O–H), 2925, 2853 (C–H) cm-1. 1H NMR (300 MHz,
CD3OD) d 0.88–1.00 (m, 6H), 1.28–1.44 (m, 36H) 2.62–2.73 (m,
2H), 2.74–2.82 (m, 1H), 3.56–3.72 (m, 3H), 3.77–3.84 (m, 1H).13C
NMR (75 MHz, CD3OD) d 13.1, 13.0, 22.4, 22.3, 25.7, 26.7, 29.1,
29.3, 29.4, 29.5, 29.6, 30.4, 31.6, 31.7, 47.9, 60.9, 63.2, 71.0, 71.7.
8168 | Org. Biomol. Chem., 2011, 9, 8163–8170
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N-((2S,3S,4R)-1,2,3-Trihydroxyoctadecan-4-yl)octanamide (ent-
16). Compound ent-16 was prepared in the same way as its
enantiomer. It displayed identical analytical data expected for its
2 (a) Y. A. Hannun and L. M. Obeid, Nat. Rev. Mol. Cell Biol., 2008,
9, 139–150; (b) N. Bartke and Y. A. Hannun, J. Lipid Res., 2009,
50(Suppl.), S91–96.
3 For reviews see: (a) J. A. Morales-Serna, J. Llaveria, Y. Diaz, M. I.
Matheu and S. Castillon, Curr. Org. Chem., 2010, 14, 2483–2521; (b) A.
R. Howell, R. C. So and S. K. Richardson, Tetrahedron, 2004, 60,
11327–11347; (c) A. R. Howell and A. J. Ndakala, Curr. Org. Chem.,
2002, 6, 365–391; (d) For recent syntheses see: Y. M. Lee, D. J. Baek,
S. Lee, D. Kim and S. Kim, J. Org. Chem., 2011, 76, 408–416; (e) Y.
M. Lee, S. Lee, H. Jeon, D. J. Baek, J. H. Seo, D. Kim and S. Kim,
Synthesis-Stuttgart, 2011, 867–872; (f) Z. Liu, H. S. Byun and R.
Bittman, J. Org. Chem., 2010, 75, 4356–4364; (g) P. Kumar, A. Dubey
and V. G. Puranik, Org. Biomol. Chem., 2010, 8, 5074–5086; (h) R.
Ait-Youcef, X. Moreau and C. Greck, J. Org. Chem., 2010, 75, 5312–
5315; (i) A. R. Parameswar, J. A. Hawkins, L. K. Mydock, M. S. Sands
and A. V. Demchenko, Eur. J. Org. Chem., 2010, 3269–3274; (j) S. J.
K. Sauerland, J. A. Castillo-Melendez, K. Nattinen, K. Rissanen and
A. M. P. Koskinen, Synthesis-Stuttgart, 2010, 757-762; (k) C. Seguin,
F. Ferreira, C. Botuha, F. Chemla and A. Perez-Luna, J. Org. Chem.,
2009, 74, 6986–6992.
25
optical rotation. [a]D -6.5 (c 1.3, MeOH).
N-((2S,3R,4R)-1,2,3-Trihydroxyoctadecan-4-yl)octanamide (17).
Aminotriol 11 (18.7 mg, 0.10 mmol) was reacted according
to the general procedure for N-acylation. The crude material
was purified by column chromatography on silica gel eluted
with EtOAc/MeOH 98 : 2 to give 17 (16.1 mg, 60%). Rf = 0.3
25
(EtOAC/MeOH 95 : 5). [a]D +10.3 (c 0.6, MeOH). IR (neat)
3405 (O–H), 2851 (C–H), 1631 (C O) cm-1. 1H NMR (300 MHz,
CD3OD) d 0.88–0.99 (m, 6H), 1.24–1.52 (m, 34H) 1.58–1.74 (m,
3
2H), 2.32 (t, J = 7.2 Hz, 2H), 3.32–3.38 (m, 1H), 3.60–3.65 (m,
3H), 3.86–3.93 (m, 1H). 13C NMR (75 MHz, CD3OD) d 14.6, 23.8
(2 peaks), 27.3 (2 peaks), 30.3, 30.4, 30.5, 30.6, 30.8 (3 peaks),
31.7, 33.0, 33.1, 37.2, 52.6, 64.4, 72.2, 74.2, 177.2. MS (ES): m/z =
444 (100%) [M + H+]. HRMS (ESI+): C26H54NO4 calc. 444.4053,
found 444.4060.
4 D. C. Phillips, S. Martin, B. T. Doyle and J. A. Houghton, Cancer Res.,
2007, 67, 756–764.
5 (a) S. Spiegel and A. H. Merrill, Jr., FASEB J, 1996, 10, 1388–1397;
(b) W. D. Jarvis, F. A. Fornari, K. L. Auer, A. J. Freemerman, E. Szabo,
M. J. Birrer, C. R. Johnson, S. E. Barbour, P. Dent and S. Grant, Mol.
Pharmacol., 1997, 52, 935–947; (c) E. R. Smith, A. H. Merrill, L. M.
Obeid and Y. A. Hannun, Methods Enzymol., 2000, 312, 361–373.
6 (a) S. Ponnusamy, M. Meyers-Needham, C. E. Senkal, S. A. Saddoughi,
D. Sentelle, S. P. Selvam, A. Salas and B. Ogretmen, Future Oncol., 2010,
6, 1603–1624; (b) A. Huwiler and U. Zangemeister-Wittke, Crit. Rev.
Oncol. Hematol., 2007, 63, 150–159.
7 (a) R. D. Baird, J. Kitzen, P. A. Clarke, A. Planting, S. Reade, A. Reid,
L. Welsh, L. L. Lazaro, B. D. L. Heras, I. R. Judson, S. B. Kaye, F.
Eskens, P. Workman, J. S. deBono and J. Verweij, Mol. Cancer Ther.,
2009, 8, 1430–1437; (b) I. Diaz-Laviada, A. M. Sanchez, S. Malagarie-
Cazenave, N. Olea, D. Vara and C. Cuevas, Eur. J. Pharmacol., 2008,
584, 237–245; (c) R. Cuadros, E. M. de Garcini, F. Wandosell, G.
Faircloth, J. M. Fernandez-Sousa and J. Avila, Cancer Lett., 2000, 152,
23–29.
8 (a) M. A. Dickson, R. D. Carvajal, A. H. Merrill, M. Gonen, L. M.
Cane and G. K. Schwartz, Clin. Cancer Res., 2011, 17, 2484–2492; (b) T.
Noda, S. Iwai, M. Hamada, Y. Fujita and Y. Yura, Apoptosis, 2009,
14, 287–297; (c) J. Coward, G. Ambrosini, E. Musi, J. P. Truman, A.
Haimovitz-Friedman, J. C. Allegood, E. Wang, A. H. Merrill and G.
K. Schwartz, Autophagy, 2009, 5, 184–193; (d) M. Hamada, T. Sumi,
S. Iwai, M. Nakazawa and Y. Yura, Apoptosis, 2006, 11, 47–56; (e) C.
W. Sachs, A. R. Safa, S. D. Harrison and R. L. Fine, J. Biol. Chem.,
1995, 270, 22859–26648; (f) G. K. Schwartz, A. Haimovitzfriedman, S.
K. Dhupar, D. Ehleiter, P. Maslak, L. Lai, F. Loganzo, D. P. Kelsen, Z.
Fuks and A. P. Albino, J. Natl. Cancer Inst., 1995, 87, 1394–1399.
9 (a) E. C. Garnier-Amblard, S. G. Mays, R. F. Arrendale, M. T. Baillie,
A. S. Bushnev, D. G. Culver, T. J. Evers, J. J. Holt, R. B. Howard,
L. S. Liebeskind, D. S. Menaldino, M. G. Natchus, J. A. Petros, H.
Ramaraju, G. P. Reddy and D. C. Liotta, ACS Med. Chem. Lett., 2011,
2, 438–443; (b) H. Symolon, A. Bushnev, Q. O. Peng, H. Ramaraju, S.
G. Mays, J. C. Allegood, S. T. Pruett, M. C. Sullards, D. L. Dillehay,
D. C. Liotta and A. H. Merrill, Mol. Cancer Ther., 2011, 10, 648–657;
(c) P. C. Roberts, N. Doyon-Reale, A. Baxa and E. M. Schmelz, FASEB
J., 2007, 21, A166–A166; (d) H. Ramaraju, A. Bushnev, C. Q. Sun, D.
C. Liotta, A. H. Merrill and J. A. Petros, J. Urol., 2008, 179, 46–46;
(e) D. S. Menaldino, A. Bushnev, A. M. Sun, D. C. Liotta, H. Symolon,
K. Desai, D. L. Dillehay, Q. Peng, E. Wang, J. Allegood, S. Trotman-
Pruett, M. C. Sullards and A. H. Merrill, Pharmacol. Res., 2003, 47,
373–381.
N - ((2S,3S,4R) - 1,3,4 - Trihydroxyoctadecan - 2 - yl)octanamide
(18). D-ribo-Phytosphingosine (18.7 mg, 0.10 mmol) was reacted
according to the general procedure for N-acylation. The crude
material was purified by column chromatography on silica gel
eluted with EtOAc/MeOH 98 : 2 to give 18 (16.1 mg, 60%). Rf = 0.3
(EtOAc/MeOH 95 : 5). [a]D +8.9 (c 1.1, MeOH). 1H NMR (300
25
MHz, CD3OD) d 0.88–0.99 (m, 6H), 1.24–1.52 (m, 34H) 1.58–1.74
3
(m, 2H), 2.32 (t, J = 7.2 Hz, 2H), 3.32–3.38 (m, 1H), 3.60–3.65
(m, 3H), 3.86–3.93 (m, 1H). 13C NMR (75 MHz, CD3OD) d =
14.5, 23.8 (2 peaks), 27.1, 27.2, 30.3, 30.4, 30.5, 30.6, 30.8, 30.9 (2
peaks), 33.0, 33.1 (2 peaks), 37.3, 53.6, 62.2, 73.4, 76.0, 176.1. MS
(ES): m/z 444 (100%) [M + H+]. HRMS (ESI+): C26H54NO4 calc.
444.4053, found 444.4051.
Biological evaluations
Cell viability experiments. Murine B16 melanoma cells were
◦
grown in a humidified 5% CO2 atmosphere at 37 C in DMEM
medium containing Glutamax (2 mM), and heat-inactivated FCS
(10%). All compounds were added to the cells as ethanolic
solution. Control cells were treated with the same concentration
of solvent (which did not exceed 0.5%). After treatment with the
compounds for 24 h in the absence of FCS, viability of murine B16
melanoma cells was evaluated by using the MTT assay based on
the cleavage of the tetrazolium salt MTT to formazan crystals by
metabolically active cells.31 The formazan crystals formed were
solubilised by adding dimethylsulfoxide for 1 h at 37 ◦C and
quantified spectrophotometrically using an ELISA reader (the
absorbance was measured at l = 560 nm).
Acknowledgements
The Pierre Fabre group and the CERPER (Centre d’Etude et de
Recherche sur la Peau et les Epithe´liums de Reveˆtement Pierre
Fabre) are gratefully acknowledged for their support and a PhD
grant to A.R.
10 M. O. Pata, Y. A. Hannun and C. K. Ng, New Phytol., 2009, 185,
611–630.
11 (a) N. Chung and L. M. Obeid, Methods Enzymol., 2000, 311, 319–
331; (b) G. M. Jenkins and Y. A. Hannun, J. Biol. Chem., 2001, 276,
8574–8581.
12 K. Nishimura, Comp. Biochem. Physiol., Part B: Biochem. Mol. Biol.,
1987, 86, 149–154.
13 P. W. Wertz, D. C. Swartzendruber, K. C. Madison and D. T. Downing,
J. Invest. Dermatol., 1987, 89, 419–425.
14 (a) J. S. Lee, D. S. Min, C. Park, C. S. Park and N. J. Cho, FEBS Lett.,
2001, 499, 82–86; (b) M. T. Park, J. A. Kang, J. A. Choi, C. M. Kang, T.
Notes and references
1 S. T. Pruett, A. Bushnev, K. Hagedorn, M. Adiga, C. A. Haynes, M.
C. Sullards, D. C. Liotta and A. H. Merrill, J. Lipid Res., 2008, 49,
1621–1639.
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©

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H. Kim, S. Bae, S. Kang, S. Kim, W. I. Choi, C. K. Cho, H. Y. Chung,
Y. S. Lee and S. J. Lee, Clin. Cancer Res., 2003, 9, 878–885; (c) M. T.
Park, J. A. Choi, M. J. Kim, H. D. Um, S. Bae, C. M. Kang, C. K.
Cho, S. M. Kang, H. Y. Chung, Y. S. Lee and S. J. Lee, J. Biol. Chem.,
2003, 278, 50624–50634; (d) Y. Nagahara, T. Shinomiya, S. Kuroda, N.
Kaneko, R. Nishio and M. Ikekita, Cancer Sci., 2005, 96, 83–92.
15 (a) S. J. Lee, M. T. Park, Y. H. Kang, I. C. Park, C. H. Kim, Y. S. Lee
and H. Y. Chung, Mol. Cancer Ther., 2007, 6, 2786–92; (b) M. T. Park,
M. J. Kim, Y. H. Kang, S. Y. Choi, J. H. Lee, J. A. Choi, C. M. Kang,
C. K. Cho, S. Kang, S. Bae, Y. S. Lee, H. Y. Chung and S. J. Lee, Blood,
2005, 105, 1724–1733.
20 H. U. Humpf, E. M. Schmelz, F. I. Meredith, H. Vesper, T. R. Vales,
E. Wang, D. S. Menaldino, D. C. Liotta and A. H. Merrill, Jr., J. Biol.
Chem., 1998, 273, 19060–19064.
21 T. Ayad, V. Faugeroux, Y. Genisson, C. Andre, M. Baltas and L.
Gorrichon, J. Org. Chem., 2004, 69, 8775–8779.
22 (a) T. Ayad, Y. Genisson, M. Baltas and L. Gorrichon, Chem. Commun.,
2003, 582–583; (b) T. Ayad, Y. Genisson and M. Baltas, Org. Biomol.
Chem., 2005, 3, 2626–2631.
23 CCDC 836021 contains the supplementary crystallographic data for
7†.
24 T. Ayad, Y. Genisson, M. Baltas and L. Gorrichon, Synlett, 2001, 866–
16 O. Hwang, G. Kim, Y. J. Jang, S. W. Kim, G. Choi, H. J. Choi, S.
Y. Jeon, D. G. Lee and J. D. Lee, Mol. Pharmacol., 2001, 59, 1249–
1255.
868.
25 CCDC 836022 contains the supplementary crystallographic data for
9†.
17 J. Esteve, A. Lorente, P. Romea, F. Urpi, C. Rios-Luci and J. M.
Padron, Eur. J. Org. Chem., 2011, 960–967; D. Mormeneo, J. Casas,
A. Llebaria and A. Delgado, Org. Biomol. Chem., 2007, 5, 3769–
3777.
18 For a review see: (a) S. Ballereau, M. Baltas and Y. Ge´nisson, Curr.
Org. Chem., 2011, 15, 953–986. For recent works see: (b) S. Lee, S. Lee,
H. J. Park, S. K. Lee and S. Kim, Org. Biomol. Chem., 2011, 9, 4580–
4586; (c) S. Ballereau, N. Andrieu-Abadie, N. Saffon and Y. Genisson,
Tetrahedron, 2011, 67, 2570–2578.
26 H. L. Kim, M. Han and D. S. Im, Prostaglandins Other Lipid Mediators,
2008, 86, 68–72.
27 SAINT-NT, Bruker AXS Inc.: Madison, Wisconsin, 2000.
28 G. M. Sheldrick, SADABS, Program for area detector adsorption
correction, Institute for Inorganic Chemistry, University of Go¨ttingen,
Germany, 1996.
29 G. M. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr., 1990,
46, 467–473.
30 G. M. Sheldrick, SHELXL-97, Program for refinement of crystal
structures, University of Go¨ttingen, Germany, 1997.
31 F. Denizot and R. Lang, J. Immunol. Methods, 1986, 89, 271–277.
19 A. Rives, S. Ladeira, T. Levade, N. Andrieu-Abadie and Y. Genisson,
J. Org. Chem., 2010, 75, 7920–7923.
8170 | Org. Biomol. Chem., 2011, 9, 8163–8170
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