Single- and double-chained truncated jaspine B analogues: Asymmetric synthesis, biological evaluation and theoretical study of an unexpected 5-endo-dig process

Article abstract of DOI:10.1016/j.tet.2011.04.027

An optimized synthesis of jaspine B analogues bearing an n-octyl or a p-fluorophenethyl lipophilic appendage was developed. Key to the approach was the use of acetylenic nucleophiles for the stereocontrolled introduction of the side chain and the implemen

Full text of DOI:10.1016/j.tet.2011.04.027

Tetrahedron 67 (2011) 4253e4262
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Single- and double-chained truncated jaspine B analogues: asymmetric synthesis,
biological evaluation and theoretical study of an unexpected 5-endo-dig process
,
b
b
c
d
d
Yahya Salma a , Stephanie Ballereau , Sonia Ladeira , Christine Lepetit , Remi Chauvin , Nathalie
ꢀ
a
b,
*
ꢀ
Andrieu-Abadie , Yves Genisson
CRCT, INSERM UMR-1037, Universite Toulouse III, CHU Rangueil, Toulouse, France
a
ꢀ
b
c
ꢀ
LSPCMIB, UMR 5068, CNRS-Universite Paul Sabatier, Toulouse, France
ꢀ
SFTCM, FR 2599, Universite Paul Sabatier, Toulouse, France
d LCC, UPR 8241, CNRS, Toulouse, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 March 2011
Received in revised form 29 March 2011
Accepted 7 April 2011
Available online 14 April 2011
An optimized synthesis of jaspine B analogues bearing an n-octyl or a p-fluorophenethyl lipophilic ap-
pendage was developed. Key to the approach was the use of acetylenic nucleophiles for the stereo-
controlled introduction of the side chain and the implementation of a novel cyclization procedure to
build the tetrahydrofuran ring. Three N-substituted amine or amide derivatives were also accessed. The
biological activity of these four jaspine B analogues was shown to strongly depend on the nature of both
the N-substituent and the aliphatic moiety connected to the tetrahydrofuran ring. Gratifyingly, the
truncated jaspine B derivative proved to be a pro-apoptotic inhibitor of the conversion of ceramide into
sphingomyelin. Finally, the efficient formation of a fused bis-furan derivative according to a 5-endo-dig
process was observed under saponification conditions. On the basis of a theoretical study, a mechanistic
pathway was delineated highlighting the Lewis acidity of the Kþ ion as the driving force for this
transformation in a strongly alkaline medium.
Keywords:
Sphingolipid
Sphingomyelin synthase
Apoptosis
5-endo-dig
Furan
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
potency. Inparticular, wedescribedtheshortchainjaspineBanalogue
3 displaying cytotoxicity towards melanoma cells comparable to that
Jaspine B (1) is an analogue of sphingolipid isolated in 2003 by
Debitus from the marine sponge Jaspis sp. (Fig 1).1 The same natural
product 1, named pachastrissamine, was also identified from extracts
of the sponge Pachatrissa sp.2 Both studies reported a potent cyto-
toxicity for compound 1, with IC50 values in the submicromolar range.
Synthetic jaspine B was found to act through a dihydroceramide-
mediated autophagy in A549 lung cancer cells.3 We evidenced that
the natural compound triggered apoptosis of melanoma cells by
blocking the conversion of ceramide into sphingomyelin.4 The am-
phiphilic structure of jaspine B embeds a phytosphingosine-like C18
skeleton. Central to its structure is the all-cis trisubstituted tetrahy-
drofuran polar head, bearing an apolar myristyl residue. Four distinct
non-natural stereoisomers of jaspine B have been disclosed to date,
indicating a variable influence of the stereochemistry on cytotoxicity,
evaluated on A549 and MCF7 breast cancer cells.3,5 Assessing the
contribution of the lipophilic portion of the molecule, we developed
a series of novel cytotoxic sphingomyelin synthase (SMS) inhibitors.6
We also found that non-amphiphilic analogues retained a substantial
of the parent natural compound.7
OH
H2N
Me
Me
O
Jaspine B (1)
NH2 OH
HO
D-ribo-Phytosphingosine (2)
OH
OH
H2N
Me
O
Truncated analogue 3
Fig. 1. Structure of the natural jaspine B (1), the natural phytosphingosine (2) and the
synthetic truncated analogue 3.
Along these lines, we wish to report here the optimized syn-
thesis and the full characterization of a series of single- and double-
* Corresponding author. Tel.: þ33 (0) 5 61 55 62 99; fax: þ33 (0) 5 61 55 60 11;
ꢀ
e-mail address: genisson@chimie.ups-tlse.fr (Y. Genisson).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.04.027

4254
Y. Salma et al. / Tetrahedron 67 (2011) 4253e4262
chained truncated jaspine B analogues, as well as a study of the
mode of action of the previously described cytotoxic derivative 3.
With the acetylenic intermediate 6 in hand, we implemented the
new cyclization protocol. The required mesylate was initially iso-
lated in 82% yield after chromatography. Due to its propensity to
undergo spontaneous cyclization, the latter could however not be
properly characterized, mass spectrometry and NMR analyses being
always ‘contaminated’ with the corresponding tetrahydrofuran. We
thus found more convenienttoengagethe crude mesylationproduct
in the cyclization step. After smooth heating in DMSO with Et3N and
BnNH2, the expected cyclized product 7 was isolated in 70% yield
overall from the alcohol 6 (84% yield based on the chromatograph-
ically purified mesylate intermediate). Even though the exact role of
both amines is not elucidated, previous studies demonstrated that
the process was more efficient in their presence.6
In order to facilitate the oxazolidinone saponification, the
nitrogen atom in 7 was first deprotected. Oxidative cleavage of the
p-methoxybenzyl group proceeded uneventfully to give the expec-
ted intermediate 8 in 77% yield. At this stage we explored the pos-
sibility of preparing the unsaturated analogue of 3. Saponification of
the acetylenic intermediate 8 led, however, to the highly efficient
formation of an unexpected product. Although the mass spectrom-
etry analysis displayed an [MþHþ] ion at m/z 212 that could account
for the expected product, the NMR analysis did not match with the
desired structure. In particular, the appearance of a strongly
unshielded quaternary 13C at 164.9 ppm and the persistence of three
1H signals above 4 ppm were puzzling. Extensive 2D NMR analysis
led us to propose the bicyclic structure 9 (Scheme 3).
2. Synthetic studies
2.1. Synthetic approach
Our previous synthesis7 of 3 starting from the aldehyde 4 relied
on two key steps: (1) the diastereoselective addition of an organo-
cerium reagent in order to introduce the lipophilic chain and (2) the
construction of the tetrahydrofuran core by means of a benzylic
ether hydrogenolysis-induced intramolecular cyclization of
a mesylate intermediate (Scheme 1).8,9,10e18 Despite of being
straightforward, this route suffered from several weak points, such
as (1) the moderate efficiency of the aliphatic organometallic addi-
tion onto the aldehyde; (2) the use of an hydrogen-promoted cy-
clization process and (3) the requirement of harsh Birch reaction
conditions in order to achieve the N-deprotection.
O
O
Organocerium addition
Mesylation
O
O
BnN
BnN
H
Me
50%
OMs
Hydrogenolysis/cyclization
O
BnO
75%
BnO
4
O
Birch debenzylation
Saponification
O
Me
O
5
BnN
3
O
O
Me
Me
5
(29% overall)
79%
Scheme 1. Previous synthetic route to 3.7
HN
O
a
H2N
H
8
O
9
O
Scheme 3. The unexpected 5-endo-dig cyclization. Reagents and conditions: (a) KOH,
In light of our recent synthetic development, we sought of op-
timizing this reaction sequence. In particular, our plan was to start
from the second-generation N-p-methoxybenzyl-protected pivotal
aldehyde 5 and to take advantage of the new convenient amine-
promoted cyclization protocol.6
EtOH/H2O (8:2), 85 ꢀC, 7 h, 92%.
The high efficiency of this unexpected transformation prompted
us to explore its mechanism by means of a theoretical study (vide
infra).
In order to ensure access to 3, saturation of the side chain in 7
was thus performed affording 10 (Scheme 4). Smooth cleavage of
the N-p-methoxybenzyl group to give 11 was followed by a final
saponification delivering the expected aminoalcohol 3 in high yield.
2.2. Preparation of truncated aliphatic analogue 3
The synthetic sequence leading to 3 started from aldehyde 5. In
order to improve the efficiency of the introduction of the lipophilic
chain, we switched from an aliphatic to an acetylenic residue. This
was also expected to favor the cyclization process onto the acti-
vated propargylic center (vide infra). Thus, treatment of aldehyde 5
with the preformed 1-octyne-derived organocerium reagent led to
the secondary aminoalcohol 6, isolated in 87% yield as a single di-
astereoisomer (Scheme 2).
O
O
O
O
Me
5
PMBN
PMBN
a
Me
5
7
O
10
O
O
O
b
OH
O
O
O
HN
c
H2N
O
Me
O
Me
5
Me
PMBN
a
PMBN
O
3
11
H
Scheme 4. The oxazolidinone hydrolysis leading to 3. Reagents and conditions: (a)
Pd(OH)2, MeOH/EtOAc (1:1), 1 bar H2, rt, 24 h, 98%; (b) CAN, MeCN/H2O (9:1), rt, 4 h,
78%; (c) KOH, EtOH/H2O (8:2), 85 ꢀC, 7 h, quant.
OH
6
O
BnO
BnO
O
5
b, c
O
The optimized five-step synthetic sequence thus led to 3 in 46%
overall yield from the pivotal aldehyde 5.
O
O
Me
Me
5
5
HN
PMBN
d
8
7
2.3. N-Functionalized aliphatic analogues 12, 13, and 14
O
O
Scheme 2. En route to 3. Reagents and conditions: (a) (i) CeCl3, THF, rt, 16 h; (ii) n-BuLi,
1-octyne, THF, 0 ꢀC, 1 h; (iii) aldehyde 5, THF, ꢁ78 ꢀC, 7 h, 87% yield, 100% de; (b) MsCl,
Et3N, CH2Cl2, 0 ꢀC to rt, 1 h, 82%; (c) BnNH2, Et3N, DMSO, 80 ꢀC, 4 h, 84%; (d) CAN,
MeCN/H2O (9:1), rt, 4 h, 77%.
We then explored the N-functionalization of the truncated jas-
pine B analogue 3. Indeed, the N-substituent of sphingolipids, by
influencing the interaction of these lipids with membranes, largely

Y. Salma et al. / Tetrahedron 67 (2011) 4253e4262
4255
orientates their cellular outcome.19e23 For instance, N-acylation of
the somewhat water-soluble sphingosine with a fatty acid leads to
ceramide, that is, confined to lipid bilayers, the synthetic N-acetyl
analogue (known as ‘C2eceramide’) being on the contrary cell per-
meable. The overall biological effect of sphingolipids is therefore
finely modulated by their N-functionalization. For instance, whereas
sphingosine is a substrate of sphingosine kinases,24 the N,N-di-
methyl sphingosine is a potent competitive inhibitor of the isoform
1 of the kinase.25 Despite early reports of naturally occurring
anhydrocerebrins, identified as 1,4-anhydrophytosphingosines
N-acylated with cerebronic (2-hydroxytetracosanoic) acid,26 the
N-functionalization of the jaspine B has merely never been studied
to date. The C12 skeleton of 3, in minimizing the amphiphilic char-
acter of the molecule, appeared appropriate for the introduction of
a second truncated lipophilic moiety.
aromatic ring-ended lipophilic appendage, such as
rophenethyl radical, embedding the same total number of carbon
atom.
The targeted compound was prepared from the acetylenic
building block 15, obtained in four steps and 44% overall yield from
aldehyde 5 (Scheme 6).6 Sonogashira coupling with 4-fluoro-
iodobenzene proceeded efficiently to afford the arylation product
16 in 70% yield. After catalytic hydrogenation, the structure of the
saturated intermediate 17 was confirmed by X-ray diffraction
analysis of a crystalline sample (Fig. 2).
a
p-fluo-
F
O
O
O
O
Ref. 6
(44%)
PMBN
a
PMBN
5
Introduction of an N-alkyl group was envisioned by mean of
a reductive amination reaction. Thus, treatment of 3 with an excess
of formaldehyde in the presence NaBH3CN gave the N,N-dimethyl
derivative 12 (Scheme 5). The N-hexyl analogue 13, comprising the
same overall length of lipophilic chain as the parent jaspine B (C14),
was also obtained from the reaction with n-hexanal.
15
O
16
O
O
b
O
F
F
OH
O
c, d
PMBN
H2N
19
17
O
Scheme 6. Synthesis of the aromatic analogue 17. Reagents and conditions: (a) IC6H4F,
PdCl2(PPh3)2, CuI, (i-Pr)2NH, THF, rt, 4 h, 70%; (b) Pd(OH)2, MeOH/EtOAc (1:1),1 bar H2, rt,
24 h, 96%; (c) CAN, MeCN/H2O (9:1), rt, 4 h, 93%; (d) KOH, EtOH/H2O (8:2), 85 ꢀC, 7 h, 96%.
NH
NH
OH
Saponification
O
O
O
O
O
KOH
O
K
Me
Me
endo-8'
Instable
hydroxy-orthocarbamate
adduct
NH
O
NH
OH
OH
O
O
O
O
K
O
K
Me
Me
endo-8'b
alcohol-carbamate
endo-8'a
alkoxide-carbamic acid
Fig. 2. Molecular view of the oxazolidinone 17 in the solid state (thermal ellipsoids at
Intramolecular
nucleophilic attack
of alkoxide
50% probability); hydrogen are omitted for clarity excepted on asymmetric carbons.27
Importantly, this structure further confirmed the all-cis ar-
rangement of the three substituents present on the tetrahydrofuran
ring. Finally, a high yielding two-step deprotection sequence de-
livered the expected jaspine B analogue 19.
Protonation /
decarboxylation
NH2
NH
O
O
O
OH
O
O
H
K
Me
Me
RO
H
3. Biological studies
Final product
endo-9'
Primary product
endo-9'a
3.1. Cell viability assays
Scheme 5. N-Functionalization of 3. Reagents and conditions: (a) aq HCHO, NaBH3CN,
AcOH, MeCN/H2O (3:1), rt, 24 h, 43%; (b) n-hexanal, NaBH3CN, MeOH, rt, 20 h, 75%; (c)
p-nitrophenyl caprilate, THF, rt, 48 h, 63%.
The lethal effect of 3 and its N-functionalized derivatives 12, 13
and 14 were evaluated on tumor cell growth and compared to that
obtained with the natural jaspine B. As illustrated in Fig. 3, the
single-chained truncated compound 3 exhibited a dose-dependent
cytotoxic activity, leading to a reduction of 50 % in cell viability of
In order to assess the influence of the basic nitrogen atom, we
also prepared an amide derivative. The N-octanoyl derivative 14
was obtained reacting 3 with p-nitrophenyl caprilate (Scheme 5).
B16 melanoma cells after 24 h exposure to 2.5 mM. Derivative 3 was
somewhat less effective than the natural molecule bearing an ali-
phatic C14 skeleton but displayed a higher or similar cytotoxicity
than its double-chained analogues 13 and 14. The presence of an
alkyl chain in the N-hexyl compound 13 led to a decrease of cyto-
toxicity compared to the N-octanoyl analogue 14. Conversely, the
dimethylation of the amine in 3 (compound 12) leads to a complete
2.4. Preparation of truncated aromatic analogue 19
We previously reported several aromatic ring-containing chain-
modified jaspine B analogues displaying potent cytotoxic effects and
SMS inhibitory activities.6 In the context of the present study, we
envisioned the replacement of the n-octyl chain in 3 by a short
loss of cytotoxicity, even at concentrations above 10
mM. The

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Y. Salma et al. / Tetrahedron 67 (2011) 4253e4262
replacement of the n-octyl chain in 3 by a short aromatic ring-
ended chain (compound 19) also abolished all cytotoxic effect at
the concentrations used. Altogether, these results suggest that,
while the length of the aliphatic chain of jaspine B has a moderate
effect on its cytotoxicity, the nature of the N-substituent and the
aliphatic portion of the molecule can strongly impact its biological
activity.
SM. As illustrated in Fig. 4D, the truncated analogue 3 elicited
a decrease in SMS activity. All these effects were comparable to that
observed with the natural jaspine B.
4. Theoretical studies
Saponification of the acetylenic intermediate 8 led to 9 via the
formation of the dihydrofuran ring that would result from an
intramolecular attack of the transient potassium alkoxide onto the
triple bond, according to a 5-endo-dig process (Scheme 3).28 Such
a pathway, believed to be favored by proper orbital overlaps, is in
agreement with the Baldwin’s rules. However, its high efficiency in
the absence of any apparent acidic activation of the acetylenic
moiety seemed surprising. This serendipitous observation promp-
ted us to seek for mechanistic insights with the help of a theoretical
study of this cyclization process.
Thorough examination of the literature indicated that formation
of a dihydrofuran via the intramolecular cyclization of a homo-
propargylic alcohol under alkaline conditions had been only spo-
radically reported. After the pioneering study by Holand and
Epsztein,29 similar transformations were either used as synthetic
steps30,31 or observed as side reactions.32 Recently, an example
related to ours, yet relying on the promoting effect of a fluorine
atom at the propargylic position, was described.33 It was note-
worthy to us that all these examples concerned homopropargylic
Fig. 3. Effect of natural jaspine B, single- and double-chained truncated analogues on
B16 melanoma cell viability.
3.2. Study of the biological effects of the truncated analogue 3
The ability of the single-chained aliphatic truncated jaspine B
analogue 3 to induce apoptotic cell death and to alter sphingolipid
metabolism was evaluated in B16 melanoma cells. Indeed, we
previously observed that the natural anhydrophytosphingosine
was able to induce apoptosis of melanoma cells by acting on the
conversion of pro-apoptotic ceramide into sphingomyelin (SM),
through the inhibition of SMS1 activity.4 Here, we reported that
executioner caspase activity, a marker of apoptosis, measured by
the cleavage of the fluorogenic tetrapeptide substrate DEVD-AMC,
increased in melanoma cells within 6 h, peaking at 16 h after
treatment with compound 3 (Fig 4A). Activation of caspase-3, i.e.,
the appearance of cleaved forms, was further demonstrated by
Western blot analysis (Fig. 4B).
alcohol substrates bearing an oxy group at the
-position31,32 with respect to the attacked triple bond.
As in the above examples, the feasibility of the 5-endo-dig
a b
-,30 -,29,33 or
g
conversion of 8 to 9 could be attributed to a specific role of the
alkaline metal cation in electrophilic activation of the alkynyl
substituent towards the homopropargylic alcoholic terminus. The
K
þ ion would thus play the role of the ‘a priori required acid in the
overall strongly basic medium’. This hypothesis was examined
through DFT calculations on a model compound 80 of 8 where the
fatty alkyl substituent of the triple bond was restricted to a methyl
Fig. 4. Effect of the single-chained truncated compound 3 on caspase activation and sphingolipid metabolism in B16 melanoma cells.
After labelling B16 cells with radioactive sphingosine, we ob-
served that the sphingolipid pattern was perturbed in cells treated
by analogue 3 as compared to untreated cells. Interestingly, the
ceramide content has strongly increased whereas SM level has
decreased by 60 % (Fig. 4C). In sharp contrast, no variation was
observed for the cellular content of glucosylceramide (GlcCer). As
inhibition of SMS activity can account for the changes observed in
the levels of SM and ceramide, we measured the in situ SMS activity
by incubating intact living cells with a fluorescent analogue of
ceramide (C6eNBDeceramide), that is, converted into fluorescent
group. Most of the calculations were performed taking into ac-
count a water medium within SCRF calculations (see computa-
tional details) as a model of the experimental ethanol/water
solvent mixture.
The oxygen atom of the starting all-cis tetrahydrofuran ring may
be pointing either towards or away from the oxazolidinone ring
(Fig. 5). At the B3PW91/6-31G** level of calculation, the respective
endo and exo conformations of 80 are isoenergetic. Both conformers
were thus considered as possible reactants in the various reaction
pathways investigated.

Y. Salma et al. / Tetrahedron 67 (2011) 4253e4262
4257
80a was calculated to be less stable by 19.1 kcal/mol than its alcohol/
carbamate tautomer endo-80b (Table S1, Scheme 7). Starting from
the exo-80 conformer, a similar situation was found, albeit at slightly
higher energies (Table S1, Fig. 6)
0
ꢁ
Fig. 5. Isoenergetic endo and exo conformations of 8 . Bond distances are given in A.
B3PW91/6-31G** level of calculation.
Since the presence of oxy groups in the vicinity of the reacting
site seemed to be required from the literature (vide supra), the
computational studies focused on a cyclization step directly ensu-
ing from the initial saponification of the oxazolidinone 80, thus
yielding a primary carbamic acid 90 (Scheme 7). Although unlikely
in view of the present study, the participation of amino alkoxide
intermediates could not be formally excluded. Subsequent pro-
tonation and decarboxylation of 90 was expected to yield the final
fused bis-furan derivative 9.
NH
NH
Fig. 6. Calculated structures and Gibbs energies of endo and exo conformations of the
OH
Saponification
O
0
0
ꢁ
O
intermediates 8 a and 8 b in aqueous medium. Bond distances are given in A. SCRF
calculations (PCM,
¼78.3553) performed at the B3PW91/6-31G** level.
O
O
O
3
KOH
O
K
Me
Me
endo-8'
Instable
In the calculated structures of 80a and 80b, as drawn in
hydroxy-orthocarbamate
adduct
Scheme 7, the Kþ/Oꢁ distances ranging from 2.6 to 2.7 A
ꢁ
(Fig. 6) were indicative of strong KeO bonds with partial ionic
character. Coordination of the potassium cation to other oxygen
atoms was indicated by still short Kþ/O distances lying in the
NH
O
NH
ꢁ
2.8e2.9 A range. Electrophilic activation of the C^C bond by the
OH
potassium cation was revealed by rather short K/Csp distances
OH
O
O
O
ꢁ
ranging from 3.3 to 3.7 A, which are shorter than a non-bonding
O
K
O
þ
ꢁ
K
K /C contact distance (3.73 A) and much shorter than the sum
Me
Me
of van der Waals radii of the corresponding neutral atoms
endo-8'b
alcohol-carbamate
endo-8'a
alkoxide-carbamic acid
ꢁ
(4.45 A).
In endo-80a and endo-80b, coordination of the tetrahydrofuran
oxygen atom to Kþ was likely to be responsible for a stabilization
with respect to the exo-isomers, wherein this oxygen atom is
located far away from the potassium centre (e.g., by 3.8 kcal/mol
for endo-80b vs exo-80b). Whereas the triple bond was symmet-
Intramolecular
nucleophilic attack
of acoholate
Protonation /
NH2
NH
O
rically ‘h
2-coordinated’ in the exo isomer, the additional O/K
decarboxylation
O
O
OH
O
bond of the endo isomer shifted the potassium cation closer to
the internal Csp atom, thus repelling it away from the external
Csp atom. This desymmetrization would make the distal Csp
atom regioselectively more electrophilic and thus more prone to
undergo nucleophilic attack by the homopropargylic oxygen
atom.
O
H
K
Me
Me
RO
H
Primary product
Final product
endo-9'
endo-9'a
Scheme 7. Structure of the saponification product of endo-80 and possible tautomeric
forms resulting from the initial hydrolysis of the oxazolidinone ring anticipated from
Starting from either the endo or exo conformations of 80a and
80b, four reaction pathways were investigated. All the processes
were found endothermic and the corresponding Gibbs free energy
profiles are shown in Fig. 7. The highest energy barriers (ca.
35e40 kcal/mol) were obtained for the most stable exo and endo
tautomers 80b, and the energy barrier was lower for the endo iso-
mer in agreement with the structural analysis of the reactants
(Table 1). In contrast, the lowest energy barriers obtained from the
less stable tautomers 80a were almost identical for both the endo
and exo conformers (ca. 20 kcal/mol).
SCRF calculations performed in aqueous medium (PCM,
31G** level.
3
¼78.3553) at the B3PW91/6-
The primary ‘hydroxy-orthocarbamate’ adduct (Scheme 7)
expected from the saponification of endo-80, could not be isolated
from SCRF calculations as a minimum on the PES. Two tautomers
80a,b resulting from the opening of oxazolidinone ring were
obtained instead. The alkoxideecarbamic acid intermediate endo-

4258
Y. Salma et al. / Tetrahedron 67 (2011) 4253e4262
Fig. 7. Computed Gibbs energy profiles of the determining steps of the KOH-induced
5-endo-dig cyclization of 80 in aqueous medium. SCRF calculations (PCM,
performed at the B3PW91/6-31G** level.
3
¼78.3553)
Fig. 8. Calculated structures of endo and exo transition states in aqueous medium.
CH3eCspeO angles are given in parentheses. Bond distances are given in A. SCRF
Table 1
ꢁ
Gibbs energies (DG) and Gibbs energy barriers (D
G#) (in kcal/mol at 298.15 K) of the
calculations (PCM,
3
¼78.3553) performed at the B3PW91/6-31G** level.
reaction pathways shown in Fig. 4. SCRF calculations (PCM,
the B3PW91/6-31G** level
3¼78.3553) performed at
Reactant
D
G#
DG
5. Conclusion
endo-80a
exo-80a
endo-80b
exo-80b
20.19
19.45
34.49
41.32
3.57
11.96
27.63
34.29
An optimized preparation of the C12 jaspine B analogue 3 was
accomplished relying on a new cyclization process to build the tet-
rahydrofuran core. Structural variations of the jaspine B were also
explored through the synthesis of four other single or double-
chained analogues. Biological evaluations of the prepared
analogues showed that, even though varying the nature of the
N-substituent or the lipophilic chain could not enhance the cyto-
toxicity, the truncated analogue of reference, 3, displayed a charac-
terized pro-apoptotic behavior towards B16 melanoma cells. Finally,
an unexpected, yet highly efficient, 5-endo-dig cyclization was
observed in the course of the saponification of an oxazolidinone
intermediate. On the basis of theoretical calculations, a mechanistic
scenario was elaborated that emphasized the key role of the po-
tassium cation in enhancing the electrophilicity of the acetylenic
partner.
According to the CurtineHammett principle, the kinetically fa-
vored cyclization pathway would thus proceed through the 80a
tautomer and involve the most nucleophilic homopropargylic ox-
ygen atom, namely an alkoxide. Although slightly less stable than
the endo-80a conformer by 1.9 kcal/mol (Table S1), the most effec-
tive reacting conformer was exo-80a (albeit by only a 0.74 kcal/mol
difference in Gibbs energy barrier). The corresponding absolute
Gibbs energy barrier of 19.5 kcal/mol is compatible with the ex-
perimental conditions (vide supra).
The calculated structures of the transition states are shown in
Fig. 8, whereas those of the primary cyclization products 90a,b are
given in Supplementary data (Fig. S1).
In agreement with the Hammond postulate, the four endo-
thermic processes involved late transition states (Fig. 8), whose
structures were indeed very close to those of the corresponding
primary products (Fig. S1). The CH3eCspeO angles deviated by ca.
15ꢀ from the ideal 120ꢀ value of the Baldwin rules,28 but the normal
mode related to the imaginary frequency involved mainly a short-
ening of the OeCsp bond, as expected. Starting from the endo-80b
reactant, the reaction coordinate indeed combined a shortening of
the OeCsp bond and a proton transfer from the reacting hydroxyl to
the nitrogen atom of the carbamate moiety.
Finally, calculations indicated that, in the absence of a potas-
sium cation, the bicyclic structure of 80 is retained upon addition
of OHꢁ to the carbamate carbonyl group. The resulting anion is
however unlikely to occur because of its high calculated energy as
compared to those of endo-80a and endo-80b. These results showed
that the potassium cation would play the determining role in the
5-endo-dig cyclization of homopropargylic alcohol derivatives
under basic conditions, in enhancing the electrophilic character of
the C^C bond. The Lewis acidity of the Kþ ion could therefore be
considered as the driving force of the process in a strongly alkaline
medium.
6. Experimental section
6.1. General details
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% phosphomolybdic acid in EtOH or
Dragendorff reagent. Column chromatographies were carried out
with SDS 35e70 mm flash silica gel. NMR spectroscopic data were
obtained with Bruker Advance 300. Chemical shifts are quoted in
parts per million (ppm) relative to residual solvent peak. J values
are given in hertz. For matter of homogeneity, sphingolipid num-
bering is used for NMR assignment throughout the experimental
section. Infrared (IR) spectra were recorded on a PerkineElmer FT-
IR 1725X spectrometer. Mass spectrometry (MS) data were
obtained on
a ThermoQuest TSQ 7000 spectrometer. High-

Y. Salma et al. / Tetrahedron 67 (2011) 4253e4262
4259
resolution mass spectra (HRMS) were performed on a Thermo-
Finnigan MAT 95 XL spectrometer. Optical rotations were measured
on a PerkineElmer model 241 spectrometer. Crystallographic data
were collected at low temperature (180 K) on a Bruker Kappa Apex
m, H4), 4.48 (2H, ABq, 2Jgem 11.8, Dd 40.4 Hz, OCH2Ph), 4.47 (1H, dd,
3J 8.1, 5.4 Hz, H3), 3.94 (1H, d, 2Jgem 15.0 Hz, NCH2Ph), 3.88e3.64 (6H,
3
5
m, OCH3, 2ꢃH1, H2), 2.17 (2H, td, JH7eH8 7.1, JH7eH4 1.9 Hz, H7),
1.54e1.41 (2H, m, 2ꢃH8), 1.40e1.18 (6H, m, 2ꢃH9, 2ꢃH10, 2ꢃH11),
II diffractometer using a graphite-monochromated Mo K
a
radiation
0.87 (3H, t, 3J 7.0 Hz, Me); 13C NMR (75 MHz, CDCl3)
d 159.3 (Cq, Ph),
ꢁ
(l¼0.71073 A) and equipped with an Oxford Cryosystems Cryo-
157.6 (C]O), 136.6 (Cq, Ph), 129.5, 128.7, 128.4, 128.3 (CH, Ph), 127.9
(Cq, Ph), 114.2 (CH, Ph), 88.1 (C5), 77.7 (C3), 77.2 (C6), 73.7 (OCH2Ph),
65.2 (C1), 61.2 (C4), 55.8 (C2), 55.3 (OCH3), 45.9 (NCH2Ph), 31.4, 28.7,
28.4, 22.6, 18.8 (5ꢃCH2, C7 to C11), 14.1 (CH3); MS (DCI/CH4) m/z 466
[MþH]þ, 100%; 494 [MþC2H5]þ, 56%; HRMS (CI) m/z C28H36NO5
requires 466.2593; found 466.2601.
stream cooler device. The structure was solved by direct methods34
and all non hydrogen atoms were refined anisotropically using the
least-squares method on F2.35 Natural jaspine B was purified from
crude extracts of the sponge Jaspis sp. kindly provided by Dr C.
Debitus (UMR 152 IRD, Toulouse, France). DMEM, trypsineEDTA
and fetal calf serum (FCS) were from Invitrogen (Cergy-Pontoise,
France). 3-(4,5-Dimethylthiazol-2-yl)-5-diphenyltetrazolium bro-
mide (MTT) was supplied from Euromedex (Mundolsheim, France).
C6eNBDeceramide was purchased from Interchim (Montluc¸ on,
France). Murine B16 melanoma cell line was purchased from
American Type Culture Collection (LGC, Molsheim, France).
6.3.2. (3aS,6S,6aS)-3-(4-Methoxybenzyl)-6-(oct-1-ynyl)tetrahy-
drofuro[3,4-d]oxazol-2(3H)-one (7). To solution of alcohol
(210 mg, 0.45 mmol) in anhydrous CH2Cl2 (5 mL) at 0 ꢀC under
nitrogen atmosphere was added Et3N (156 L, 1.12 mmol). After
15 min of stirring at the same temperature mesyl chloride (190 L,
6
m
m
1.12 mmol) was added and the solution was stirred at 0 ꢀC for
30 min then at room temperature for 1 h. The reaction was
quenched by addition of water and the mixture was extracted three
times with CH2Cl2. The combined extracts were washed with brine,
dried over Na2SO4, and concentrated in vacuo. The crude material
was purified by column chromatography on SiO2 eluted with PE/
EtOAc (70:30 to 60:40) to give the expected mesylate (200 mg, 82%)
as a colorless oil. However, this intermediate gave rise to sponta-
neous cyclization and was thus routinely used without purification
for the next step.
6.2. General chemical procedures
6.2.1. General procedure A: oxidative cleavage of the p-methoxy-
benzyl group. To a solution of N-PMB intermediate (1.0 equiv) in
CH3CN/H2O (9:1) (0.05 M) was added CAN (6.0 equiv). The mixture
was stirred at room temperature for 2 h, then diluted with water
and extracted three times with EtOAc. The combined organic layers
were washed with saturated aqueous NaHCO3 solution and with
brine, dried over MgSO4, and concentrated in vacuo.
To a solution of the chromatographically purified mesylate
(195 mg, 0.36 mmol) in anhydrous DMSO (6 mL) at room temper-
6.2.2. General procedure B: saponification of the carbamate. KOH
(10 equiv) was added to a solution of oxazolidinone (1.0 equiv) in
EtOH/H2O (8:2) (0.05 M). The mixture was heated at 85 ꢀC for
7e10 h (TLC monitoring) before being cooled to room temperature
and diluted with EtOAc and brine. The mixture was extracted three
times with EtOAc and the combined extracts were concentrated in
vacuo.
ature and under nitrogen atmosphere were added Et3N (150
mL,
1.08 mmol) and BnNH2 (59.0 L, 0.54 mmol). The solution was
m
heated at 80 ꢀC with stirring for 4 h. The mixture was then diluted
with CH2Cl2 and the reaction quenched by addition of water. The
mixture was extracted three times with CH2Cl2. The combined or-
ganic layers were washed with water and brine, dried over MgSO4,
and concentrated in vacuo. The crude material was purified by
column chromatography on SiO2 eluted with PE/EtOAc (60:40) to
give 7 (108 mg, 84%) as a white solid. This sequence was run
without purification of the intermediate mesylate with equivalent
6.2.3. General procedure C: hydrogenation of the triple bonds. A
solution of unsaturated compound (1.0 equiv) in EtOAc/MeOH (1:1)
(0.1 M) containing Pd(OH)2 (20 wt %) was stirred under H2 at at-
mosphere pressure (balloon) overnight. The reaction mixture was
then filtered over Celite. The precipitate was rinsed with CH2Cl2 and
the filtrate was concentrated in vacuo.
efficiency (i.e., 70% overall yield). ½a D20
ꢂ
þ105.2 (c 1.2, CHCl3); IR
(film) nC
] ; d
O 1740 cmꢁ1 1H NMR (300 MHz, CDCl3) 7.18 (2H, d, 3J
8.6 Hz, NCH2Ph), 6.86 (2H, d, 3J 8.6 Hz, NCH2Ph), 4.80 (1H, dd,
3JH3eH2 7.6, 3JH3eH4 4.2 Hz, H3), 4.65 (1H, d, 2Jgem 15.0 Hz, NCH2Ph),
4.34e4.27 (1H, m, H4), 4.11 (1H, d, 2Jgem 15.0 Hz, NCH2Ph), 4.07 (1H,
6.3. Specific chemical procedures and characterization data
3
3
2
0
dd, JH2eH3 7.9, JH2eH1 4.0 Hz, H2), 3.91 (1H, d, Jgem 10.6 Hz, H1),
0
0
6.3.1. (4S,5S)-4-(Benzyloxymethyl)-5-((R)-1-hydroxynon-2-ynyl)-3-
(4-methoxybenzyl) oxazolidin-2-one (6). A suspension of anhydrous
CeCl3 (0.86 g, 3.52 mmol) in anhydrous THF (10 mL) under nitrogen
atmosphere was stirred overnight at room temperature. The sus-
pension of CeCl3 was cooled to ꢁ78 ꢀC and a freshly prepared so-
lution of octynyl lithium generated by addition of n-BuLi (2.65 mL
of a 1.6 M commercial solution in hexanes, 4.20 mmol) to 1-octyne
3.85 (3H, s, OCH3), 3.36 (1H, dd, 2Jgem 10.6, 3JH1 eH2 4.0 Hz, H1 ), 2.24
3
5
(2H, td, JH7eH8 7.1, JH7eH4 1.8 Hz, H7), 1.60e1.46 (2H, m, H8),
1.44e1.18 (6H, m, 3ꢃCH2, H9 to H11), 0.87 (3H, t, 3J 6.7 Hz, Me); 13
C
NMR (75 MHz, CDCl3)
d 159.4(Cq, Ph), 157.0 (C]O), 129.5 (CH, Ph),
127.1 (Cq, Ph), 114.2 (CH, Ph), 90.5 (C5 or C6), 77.1 (C3), 73.6 (C4), 71.3
(C6 or C5), 69.3 (C1), 59.4 (C2), 55.2 (OCH3), 46.2 (NCH2Ph), 31.2,
28.4, 28.2, 22.4, 18.8 (C7 to C11), 13.9 (Me); MS (ESI) m/z 380
[MþNa]þ, 100%; HRMS (CI) m/z C21H27NO4Na requires 380.1838;
found 380.1858.
(620
m
L, 4.20 mmol) in anhydrous THF (10 mL) at ꢁ20 ꢀC was added
dropwise. The reaction mixture was stirred at ꢁ78 ꢀC for 1 h. Then
aldehyde 5 (250 mg, 0.70 mmol) in solution in anhydrous THF
(2 mL) was added and the mixture stirred at ꢁ78 ꢀC for 6 h. The
reaction was quenched by addition of a saturated aqueous solution
of NH4Cl and the mixture was directly filtered over Celite eluted by
EtOAc. The aqueous layer was extracted with EtOAc. The combined
extracts were washed with brine, dried over Na2SO4, and concen-
trated in vacuo. The crude material was purified by column chro-
matography on SiO2 eluted with PE/EtOAc (70:30 to 60:40) to give 6
6.3.3. (3aS,6S,6aS)-6-(Oct-1-ynyl)tetrahydrofuro[3,4-d]oxazol-
2(3H)-one (8). Prepared from N-PMB derivative
7 (70.0 mg,
0.19 mmol) according to the general procedure A. The crude ma-
terial was purified by column chromatography on SiO2 eluted with
CH2Cl2/EtOAc (60:40 to 50:50) to give 8 (30.0 mg, 65%) as a white
solid. ½a 2D0
ꢂ
þ153.4 (c 1.4, CHCl3); IR (film) nC
]
O 1727 cmꢁ1; 1H NMR
3
(300 MHz, CDCl3)
d
6.80 (1H, ls, NH), 4.97 (1H, dd, JH3eH2 7.6,
(285 mg, 87%) as a colorless oil. ½a D20
ꢂ
þ27.8 (c 0.8, CHCl3); IR (film)
3JH3eH4 4.0 Hz, H3), 4.39 (1H, dd, 3JH2eH3 7.6, 3JH2eH1 4.2 Hz, H2), 4.30
3
5
2
nOH 3402, nC
]
O 1752 cmꢁ1; 1H NMR (300 MHz, CDCl3)
d
7.42e7.22
(1H, dt, JH4eH3 4.0, JH4eH7 1.9 Hz, H4), 3.98 (1H, d, Jgem 10.5 Hz,
2
3
(5H, m, OCH2Ph), 7.09 (2H, d, 3J 8.6 Hz, NCH2Ph), 6.81 (2H, d, 3J
8.6 Hz, NCH2Ph), 4.70 (1H, d, 2Jgem 15.0 Hz, NCH2Ph), 4.70e4.65 (1H,
H1), 3.57 (1H, dd, Jgem 10.5, JH1 eH2 4.2 Hz, H1 ), 2.24 (2H, dt,
0
0
3JH7eH8 7.1, 5JH7eH4 1.9 Hz, H7), 1.58e1.46 (2H, m, H8), 1.44e1.16 (6H,

4260
Y. Salma et al. / Tetrahedron 67 (2011) 4253e4262
3
3
m, H9 to H11), 0.86 (3H, t, 3J 6.8 Hz, Me); 13C NMR (75 MHz, CDCl3)
159.2 (C]O), 90.3 (C5 or C6), 80.3 (C3), 73.4 (C4), 73.2 (C1), 71.4 (C6
or C5), 56.7 (C2), 31.2, 28.4, 28.2, 22.4, 18.7 (C7 to C11), 13.9 (Me); MS
(DCI/NH3) m/z 260 [MþNa]þ, 100%; HRMS (CI) m/z C13H19NO3Na
requires 260.1263; found 260.1270.
H1), 3.86 (1H, dd, JH3eH2 5.0, JH3eH4 3.6 Hz, H3), 3.73 (1H, ddd,
3
3
3
3
0
d
JH4eH5 7.5, JH4eH5 6.3, JH4eH3 3.4 Hz, H4), 3.66 (1H, pseudot,
3
2
3
0
0
JH2eH1 7.0, JH2eH3 5.2 Hz, H2), 3.51 (1H, dd, JH1 eH1 8.6, JH1 eH2
0
6.8 Hz, H1 ), 1.90e1.60 (5H, m, 2ꢃH5, NH2, OH), 1.50e1.20 (12H, m,
6ꢃCH2), 0.90 (3H, t, 3J 7.0 Hz, Me); 13C NMR (75 MHz, CDCl3)
d 83.2
(C4), 72.3 (C1), 71.7 (C3), 54.3 (C2), 31.9 (C5), 29.8, 29.7, 29.5, 29.3,
26.3, 22.7 (C6 to C11), 14.1 (C12); SM (DCI/NH3) m/z 216 [MþH]þ,
62%; 233 [MþNH4]þ, 100%; HRMS (CI) m/z C12H26NO2 requires
216.1964; found 216.1965.
6.3.4. (3S,3aS,6aS)-5-Hexyl-2,3,3a,6a-tetrahydro-furo[3,2-b]furan-3-
amine (9). Prepared from oxazolidinone 8 (17.2 mg, 0.07 mmol)
according to the general procedure B. The crude material was pu-
rified by column chromatography on SiO2 eluted with EtOAc/
MeOH/NH4OH (94.2:5:0.8) to give 9 (14.0 mg, 91%) as a colorless oil.
6.3.8. (2S,3S,4S)-4-(Dimethylamino)-2-octyltetra-hydrofuran-3-ol
(12). To a solution of amine 3 (22.0 mg, 0.10 mmol) in MeCN/H2O
(3:1) (2 mL) at room temperature were added formaldehyde
½
a 2D0
ꢂ
ꢁ4.2 (c 1.3, CHCl3); IR (film) nNH 3430 cmꢁ1
;
1H NMR
3
3
(500 MHz, CDCl3)
d 5.29 (1H, dd, JH4eH3 6.2, JH4eH5 2.6 Hz, H4),
4.69 (1H, d, 3JH5eH4 2.6 Hz, H5), 4.64 (1H, pseudot,
(30.0
borohydride NaBH3CN (19.6 mg, 0.30 mmol), and acetic acid
(12.0 L, 0.20 mmol). The mixture was stirred for 24 h after which it
mL of a 37% aqueous solution, 0.40 mmol), sodium cyano-
3JH3eH4z JH3eH2z5.9 Hz, H3), 3.86 (1H, dd, JH1eH1 8.6, JH1eH2
3
2
3
0
6.7 Hz, H1), 3.45 (1H, ddd, 3JH2eH1 10.4, JH2eH1 6.7, 3JH2eH3 5.6 Hz,
m
3
0
3
2
0
0
0
H2), 3.02 (1H, dd, JH1 eH2 10.4, JH1 eH1 8.6 Hz, H1 ), 2.17 (2H, t,
3JH7eH8 7.6 Hz, 2ꢃH7), 1.60e1.44 (4H, m, 2ꢃH8, NH2), 1.40e1.20 (6H,
m, 3ꢃCH2), 0.86 (3H, t, 3J 6.8 Hz, Me); 13C NMR (75 MHz, CDCl3)
was diluted with water. The mixture was extracted three times
with CH2Cl2 and the combined extracts were washed with brine
and dried over mgSO4. The solvent was evaporated in vacuo and the
crude material was purified by column chromatography on SiO2
eluted with EtOAc/CH2Cl2/NH4OH (49.2:50:0.8) to give 12 (10.7 mg,
d
164.9 (C6), 95.5 (C5), 85.7 (C4), 83.7 (C3), 68.4 (C1), 56.1 (C2), 31.5
(C7), 28.9, 27.9, 26.7, 22.6 (C8 to C11), 14.1 (C12); SM (DCI/NH3) m/z
212 [MþH]þ, 60%; 234 [MþNa]þ, 65%; HRMS (CI) m/z C12H22NO2
requires 212.1651; found 212.1653.
a 20
43%) as colorless oil. ½ ꢂD þ36.9 (c 0.2, CHCl3); IR (film) nOH
3430 cmꢁ1
;
1H NMR (300 MHz, CDCl3)
d
3.99 (1H, dd, JH3eH2 4.0,
0
3
3
JH3eH4 2.8 Hz, H3), 3.92e3.76 (3H, m, H1 , H1, H4), 2.99 (1H, br s,
OH), 2.82 (1H, ddd, 3JH2eH1 9.2, JH2eH1 7.7, 3JH2eH3 4.1 Hz, H2), 2.31
3
0
6.3.5. (3aS,6S,6aS)-3-(4-Methoxybenzyl)-6-octyl-tetrahydrofuro[3,4-
d]oxazol-2(3H)-one (10). Prepared from alkyne
0.22 mmol) according to the general procedure C. The crude ma-
7
(80.0 mg,
(6H, br s, 2ꢃNCH3), 1.76e1.62 (2H, m, 2ꢃH5), 1.40e1.20 (12H, m,
6ꢃCH2), 0.87 (3H, t, 3J 6.7 Hz, Me); 13C NMR (75 MHz, CDCl3)
d 83.4
terial was purified by column chromatography on SiO2 eluting with
(C4), 70.4 (C3), 70.2 (C2), 66.6 (C1), 44.2 (2ꢃNCH3), 31.9 (C5), 29.8,
29.5, 29.3, 29.2, 26.2, 22.6 (C6 to C11), 14.1 (C12); MS (DCI/CH4) m/z
244 [MþH]þ, 100%; HRMS (CI) m/z C14H30NO2 requires 244.2277;
found 244.2256.
CH2Cl2/EtOAc (95:5) to give 10 (79.0 mg, 98%) as a white solid. ½a D20
ꢂ
þ70.8 (c 1.0, CHCl3); IR (film) nC
]
O 1737 cmꢁ1; 1H NMR (300 MHz,
CDCl3)
d
7.18 (2H, d, 3J 8.6 Hz, NCH2Ph), 6.86 (2H, d, 3J 8.6 Hz,
3
3
NCH2Ph), 4.74 (1H, dd, JH3eH2 7.6, JH3eH4 3.8 Hz, H3), 4.69 (1H, d,
2Jgem 15.0 Hz, NCH2Ph), 4.07 (1H, d, 2Jgem 15.0 Hz, NCH2Ph), 4.06 (1H,
6.3.9. (2S,3S,4S)-4-(Hexylamino)-2-octyltetra-hydrofuran-3-ol
(13). To a solution of amine 3 (20.0 mg, 0.09 mmol) in distilled
MeOH (1.5 mL) at room temperature and under nitrogen atmo-
sphere were added sodium cyanoborohydride NaBH3CN (11.6 mg,
3
3
2
0
dd, JH2eH3 7.6, JH2eH1 4.0 Hz, H2), 3.92 (1H, d, Jgem 10.6 Hz, H1),
3.78 (3H, s, OCH3), 3.48 (1H, td, 3JH4eH5 6.8, 3JH4eH3 3.8 Hz, H4), 3.29
2
3
0
0
(1H, dd, Jgem 10.6, JH1 eH2 4.0 Hz, H1 ), 1.82e1.68 (2H, m, 2ꢃH5),
1.50e1.16 (12H, m, 6ꢃCH2), 0.86 (3H, t, 3J 6.7 Hz, Me); 13C NMR
0.18 mmol), acetic acid (5.30 mL, 0.09 mmol), and hexanal (11.0 mL,
(75 MHz, CDCl3)
d
159.4(Cq, Ph), 157.5 (C]O), 129.5 (CH, Ph), 127.3
0.09 mmol). The mixture was stirred for 20 h after which it was
diluted with water. The mixture was extracted three times with
diethyl ether and the combined extracts were washed with brine
and dried over mgSO4. The solvent was evaporated in vacuo and
the crude material was purified by column chromatography on
SiO2 eluted with EtOAc/MeOH/NH4OH (84.2:15:0.8) to give 13
(Cq, Ph), 114.2 (CH, Ph), 83.4 (C4), 77.7 (C3), 69.2 (C1), 59.8 (C2), 55.2
(OCH3), 46.1 (NCH2Ph), 31.7, 29.5, 29.3, 29.1, 27.9, 25.9, 22.5 (C5 to
C11), 14.0 (Me); MS (DCI/CH4) m/z 362 [MþH]þ, 100%; 390
[MþC2H5]þ, 18%; HRMS (CI) m/z C21H32NO4 requires 362.2331;
found 362.2329.
(20.7 mg, 75%) as a white solid. ½a D20
ꢂ
þ22.1 (c 1.0, CHCl3); IR (film)
6.3.6. (3aS,6S,6aS)-6-Octyltetrahydrofuro[3,4-d]oxazol-2(3H)-
one(11). Prepared from N-PMB derivative 10 (55.8 mg, 0.15 mmol)
according to the general A. The crude material was purified by
column chromatography on SiO2 eluted with CH2Cl2/EtOAc (70:30
nOH 3100, nNH 3275 cmꢁ1 1H NMR (300 MHz, CDCl3)
; d 3.96e3.88
3
3
(2H, m, H1, H3), 3.69 (1H, td, JH4eH5 6.9, JH4eH3 3.0 Hz, H4), 3.51
(1H, dd, 2JH1 eH1 8.5, 3JH1 eH2 7.2 Hz, H1 ), 3.35 (1H, td, JH2eH1 7.2,
3
0
0
0
0
3JH2eH3 4.9 Hz, H2), 2.70e2.50 (2H, m, 2ꢃH5), 1.78e1.58 (2H, m,
to 50:50) to give 11 (28.8 mg, 77%) as a white solid. ½a D20
ꢂ
þ77.7 (c
2ꢃH6), 1.54e1.18 (20H, m, 10ꢃCH2), 0.92e0.80 (6H, m, 2ꢃMe); 13
C
0.6, CHCl3); IR (film) nC
]
1740 cmꢁ1
;
1H NMR (300 MHz, CDCl3)
NMR (75 MHz, CDCl3) d 83.5 (C4), 70.6 (C1), 69.6 (C3), 61.9 (C2),
O
3
3
d
5.95 (1H, br s, NH), 4.88 (1H, dd, JH3eH4 3.6, JH3eH2 7.5 Hz, H3),
48.8 (NCH2), 31.9, 31.7, 30.2, 29.8, 29.5, 29.3, 29.2, 26.8, 26.3, 22.6,
22.5 (11ꢃCH2), 14.1, 14.0 (2ꢃCH3); MS (DCI/CH4) m/z 300 [MþH]þ,
100%; HRMS (CI) m/z C18H38NO2 requires 300.2903; found
300.2892.
4.30 (1H, dd, 3JH2eH3 7.5, 3JH2eH1 3.9 Hz, H2), 3.66 (2H, AB of an ABX,
Jgem 10.5, 3JH1eH2 3.9, 3JH1 eH2 0.0 Hz, Dd 129 Hz, 2ꢃH1), 1.75e1.65
2
0
(2H, m, 2ꢃH5), 1.40e1.15 (12H, m, 6ꢃCH2), 0.81 (3H, t, 3J 7.0 Hz,
Me); 13C NMR (75 MHz, CDCl3)
d 159.0 (C]O), 83.2 (C4), 81.0 (C3),
73.3 (C1), 57.2 (C2), 31.8, 29.6, 29.4, 29.2, 28.1, 26.0, 22.6 (C5 to C11),
14.1 (Me); MS (DCI/NH3) m/z 259 [MþNH4]þ, 100%; HRMS (CI) m/z
C13H24NO3 requires 242.1756; found 242.1757.
6.3.10. N-((3S,4S,5S)-4-Hydroxy-5-octyltetra-hydrofuran-3-yl)octa-
namide (14). To a solution of amine 3 (13.7 mg, 0.06 mmol) in
anhydrous THF (1.5 mL) at room temperature and under nitrogen
atmosphere was added 4-nitrophenyl caprylate (31.0
mL,
6.3.7. (2S,3S,4S)-4-Amino-2-(octyl)tetrahydrofuran-3-ol
(3). Prepared from oxazolidinone 11 (48.0 mg, 0.22 mmol)
according to the general procedure B. The crude material was pu-
rified by column chromatography on SiO2 eluted with EtOAc/
MeOH/NH4OH (69.2:30:0.8) to give 3 (45.5 mg, quant.) as a white
0.12 mmol). The mixture was stirred for 2 days after which solvent
was removed in vacuo. The crude material was purified by column
chromatography on SiO2 eluted with PE/EtOAc (70:30) to give 14
(13.8 mg, 63%) as a white solid. ½a D20
ꢂ
þ5.3 (c 0.9, CHCl3); IR (film)
nOH,
d
3326, nNHC
]
1641 cmꢁ1
O
;
1H NMR (300 MHz, CDCl3)
NH
solid. ½a 2D0
ꢂ
þ20.0 (c 0.5, CHCl3); IR (neat) nOH, NH 3340 cmꢁ1
;
1H
6.13(1H, d, 3J 7.6 Hz, NH), 4.57 (1H, pseudoqd, 3JH2eH1 7.6, J 5.0 Hz,
3
0
2
3
3
3
0
NMR (500 MHz, CDCl3)
d
3.93 (1H, dd, JH1eH1 8.6, JH1eH2 7.5 Hz,
H2), 4.14e4.04 (2H, m, H1, H3), 3.76 (1H, td, JH4eH5 6.9, JH4eH3

Y. Salma et al. / Tetrahedron 67 (2011) 4253e4262
4261
2.8 Hz, H4), 3.57 (1H, dd, 2JH1 eH1 8.8, 3JH1 eH2 7.3 Hz, H1 ), 2.20 (2H,
pseudot, 3Jz7.6 Hz, 2ꢃH5), 1.74e1.54 (4H, m, 2ꢃCH2), 1.38e1.18
(20H, m, 10ꢃCH2), 0.94e0.80 (6H, m, 2ꢃMe); 13C NMR (75 MHz,
CDCl3) d
7.16 (2H, dd, 3J 8.5, 4J 5.5 Hz, PhF), 6.95 (2H, t, 3J 8.7 Hz, PhF),
0
0
0
6.82 (1H, br s, NH), 4.89 (1H, dd, 3JH3eH2 7.5, 3JH3eH4 3.7 Hz, H3), 4.36
2
(1H, dd, 3JH2eH3 7.5, 3JH2eH1 3.9 Hz, H2), 3.95 (1H, d, Jgem 10.4 Hz, H1),
0
0
CDCl3)
d
173.5 (C]O), 82.1 (C4), 72.0 (C3), 70.6 (C1), 52.8 (C2), 36.6,
3.54e3.4 (2H, m, H4, H1 ), 2.88e2.64 (2H, m, 2ꢃH5), 2.20e1.90 (2H, m,
31.8, 31.6, 29.7, 29.4, 29.3, 29.2, 28.9, 28.8, 26.0, 25.6, 22.6, 22.5
(13ꢃCH2), 14.1, 14.0 (2ꢃCH3); MS (DCI/CH4) m/z 342 [MþH]þ, 15%;
364 [MþNa]þ, 100%; HRMS (CI) m/z C20H40NO3 requires 342.3008;
found 342.2990.
2ꢃH6); 13C NMR (75 MHz, CDCl3)
d
161.3(d, Cq, PhF, 1J 244 Hz), 159.4
(C]O),136.7(d, Cq, PhF, 4J 3.2 Hz), 129.7 (d, CH, PhF, 3J 7.8 Hz), 115.1 (d,
CH, PhF, 2J 21.1 Hz), 81.8 (C4), 80.8 (C3), 73.3 (C1), 57.2 (C2), 31.2 (C6),
29.7 (C5); MS (DCI/CH4) m/z 252 [MþH]þ, 100%; HRMS (CI) m/z
C13H15NO3F requires 252.1036; found 252.1028.
6.3.11. (3aS,6S,6aS)-6-((4-Fluorophenyl)ethynyl)-3-(4-methoxybenzyl)-
tetrahydrofuro[3,4-d]oxazol-2(3H)-one (16). To a solution of alkyne 15
(83.0 mg, 0.30 mmol) and iodofluorobenzene (81.0 mg, 0.36 mmol) in
degassed THF (5 mL) under argon in the dark were added
6.3.14. (2S,3S,4S)-4-Amino-2-(4-fluorophenethyl)-tetrahydrofuran-
3-ol (19). Prepared from oxazolidinone 18 (30.0 mg, 0.12 mmol)
according to the general procedure B. The crude material was
purified by column chromatography on SiO2 eluted with EtOAc/
MeOH/NH4OH (84.2:15:0.8) to give 19 (25.8 mg, 96%) as a white
Pd(PPh3)2Cl2 (8.00 mg), CuI (2.30 mg), and diisopropylamine (106 mL,
0.75 mmol). The reaction mixture was stirred for 4 h at room tem-
perature and quenched with saturated NH4Cl solution. The mixture
was extracted at three times with EtOAc and the combined extracts
were washed with brine, dried over mgSO4, and concentrated in
vacuum. The crude material was purified by column chromatography
on SiO2 eluted with CH2Cl2/EtOAc (95:5) to give 16 (78.0 mg, 70%) as
solid. ½a 2D0
ꢂ
þ22.0 (c 1.1, CHCl3); IR (film) nOH
,
2937 cmꢁ1 1H
;
NH
NMR (300 MHz, CD3OD)
d
7.21 (2H, dd, 3J 8.2 Hz, 4J 5.7 Hz, PhF),
6.97 (2H, t, 3J 8.8 Hz, PhF), 3.98e3.76 (3H, m, H1, H3, H4),
0
3.55e3.38 (2H, m, H1 , H2), 2.78e2.56 (2H, m, H5), 1.90 (2H, dd,
2Jgem 14.8, 3J
7.8 Hz, H6); 13C NMR (75 MHz, CD3OD)
H6eH5
colorless oil. ½a D20
ꢂ
þ151.7 (c 0.9, CHCl3); IR (film) nC
]
7.48 (2H, dd, 3J 8.8, 4J 5.4 Hz, PhF), 7.20 (2H, d,
O 1737 cmꢁ1; 1H
d
162.7(d, Cq, PhF, 1J 242 Hz), 139.3(d, Cq, PhF, 4J 3.2 Hz), 131.0 (d,
NMR (300 MHz, CDCl3)
d
CH, PhF, 3J 7.8 Hz), 115.9 (d, CH, PhF, 2J 21.3 Hz), 83.3 (C4), 73.3 (C3),
72.2 (C1), 56.1 (C2), 32.8 (C6), 32.4 (C5); MS (DCI/CH4) m/z 226
[MþH]þ, 25%; HRMS (CI) m/z C12H17NO2F requires 226.1243;
found 226.1250.
3J 8.6 Hz, NCH2Ph), 7.00 (2H, d, 3J 8.8 Hz, PhF), 6.87 (2H, d, 3J 8.6 Hz,
3
3
NCH2Ph), 4.91 (1H, dd, JH3eH2 7.8, JH3eH4 4.4 Hz, H3), 4.66 (1H, d,
2Jgem 15.0 Hz, NCH2Ph), 4.56 (1H, d, JH4eH3 4.4 Hz, H4), 4.17 (1H, d,
3
2
3
3
0
Jgem 15.0 Hz, NCH2Ph), 4.14 (1H, dd, JH2eH3 7.7, JH2eH1 4.3 Hz, H2),
2
2
3.97 (1H, d, Jgem 10.6 Hz, H1), 3.79 (3H, s, OCH3), 3.46 (1H, dd, Jgem
10.6, 3JH1 eH2 4.3 Hz, H1 ); 13C NMR (75 MHz, CDCl3)
d
162.8(d, Cq, PhF,
6.4. Biological evaluations
0
0
1J 250.3 Hz), 159.5(Cq, Ph), 157.0 (C]O), 134.0 (d, CH, PhF, 3J 8.5 Hz),
129.5 (CH, Ph), 127.0 (Cq, Ph), 117.9 (d, Cq, PhF, 4J 3.4 Hz), 115.5 (d, CH,
PhF, 2J 21.1 Hz), 114.3 (CH, Ph), 88.0 (C5), 80.1 (C6), 77.1 (C3), 73.7 (C4),
69.7 (C1), 59.5 (C2), 55.2 (OCH3), 46.4 (NCH2Ph); MS (DCI/CH4) m/z 368
[MþH]þ, 66%; HRMS (CI) m/z C21H19NO4F requires 368.1298; found
368.1310.
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%).
6.3.12. (3aS,6S,6aS)-6-(4-Fluorophenethyl)-3-(4-methoxybenzyl)tetra-
hydrofuro[3,4-d]oxazol-2(3H)-one (17). Prepared from alkyne 16
(62.3 mg, 0.17 mmol) according to the general procedure C to give 17
6.4.1. Cell viability. After treatment with the indicated concentra-
tions of natural Jaspine B or synthetic truncated analogues for 24 h
in the absence of FCS, viability of murine B16 melanoma cells was
assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) assay.36
(60.1 mg, 95%) as a white solid. ½a D20
þ65.4 (c 1.3, CHCl3); IR (film)
ꢂ
nC
] ; d 7.24e7.10 (4H, m
1738 cmꢁ1 1H NMR (300 MHz, CDCl3)
O
containing a d, 3J 8.6 Hz, NCH2Ph, PhF), 6.95 (2H, t, 3J 8.7 Hz, PhF), 6.87
(2H, d, 3J 8.6 Hz, NCH2Ph), 4.72 (1H, dd, 3JH3eH2 7.6, 3JH3eH4 3.9 Hz, H3),
6.4.2. Caspase activation. After incubation with 5 mM of compound 3
4.69 (1H, d, 2Jgem 15.0 Hz, NCH2Ph), 4.09 (1H, d, 2Jgem 15.0 Hz, NCH2Ph),
for the indicated times in the absence of FCS, cells were sedimented
and washed with PBS. Cell pellets were homogenized in 10 mM
HEPES (pH 7.4), 42 mM KCl, 5 mM MgCl2, 0.5% CHAPS, 1 mM
4.06 (1H, dd, 3JH2eH3 7.6, 3JH2eH1 4.0 Hz, H2), 3.94 (1H, d, Jgem 10.6 Hz,
2
0
H1), 3.79 (3H, s, OCH3), 4.45 (1H, ddd, 3JH4eH5 7.6, 3JH4eH5 6.0, 3JH4eH3
0
2
3
0
0
3.9 Hz, H4), 3.28 (1H, dd, Jgem 10.6, JH1 eH2 4.0 Hz, H1 ), 2.84e2.62
dithiothreitol, 1 mM PMSF, and 2
mg/ml leupeptin. Reaction mixtures
(2H, m, 2ꢃH5), 2.20e1.94 (2H, m, 2ꢃH6); 13C NMR (75 MHz, CDCl3)
contained 100 l of cell lysates and 100
m
m
l of 40 M Ac-Asp-Glu-Val-
m
d
161.3(d, Cq, PhF, 1J 244 Hz), 159.4(Cq, Ph), 157.3 (C]O), 136.7 (d, Cq,
Asp-aminomethylcoumarin (Ac-DEVD-AMC). After 30 min in-
cubation at room temperature, the amount of the released fluorescent
product aminomethylcoumarin was determined at 351 and 430 nm
for the excitation and emission wavelengths, respectively. Protein
concentrations were determined according to the Bradford method.
Then, equal amounts of proteins were separated in a 15% SDS/
polyacrylamide gel and transferred to a nitrocellulose membrane
and blotted with monoclonal anti-caspase-3 or anti-b-actin (used
as a control for protein loading) antibodies. Proteins were detected
using an ECL detection system.
PhF, 4J 3.2 Hz), 129.7 (d, CH, PhF, 3J 7.8 Hz), 129.5 (CH, Ph), 127.2 (Cq,
Ph), 115.1 (d, CH, PhF, 2J 21.1 Hz), 114.2 (CH, Ph), 82.0 (C4), 77.5 (C3),
69.2 (C1), 59.9 (C2), 55.2 (OCH3), 46.1 (NCH2Ph), 31.1 (C6), 29.5 (C5); MS
(DCI/CH4) m/z 372 [MþH]þ, 100%; [MꢁCH3OPh]þ, 28%; [MþC2H5]þ,
15%; HRMS (CI) m/z C21H23NO4F requires 372.1611; found 372.1606.
Selected crystallographic data for x: C21H22FNO4, M¼371.4, triclinic,
ꢁ
ꢁ
ꢁ
space group P1, a¼4.6676(4) A, b¼17.2070(13) A, c¼17.7086(13) A,
3
ꢁ
ꢀ
a
¼75.915(4)ꢀ,
b
¼87.483(4)ꢀ,
¼82.706(4) , V¼1368.23(19) A , Z¼3,
g
crystal size 0.60ꢃ0.38ꢃ0.10 mm3, 29,473 reflections collected (8223
independent, Rint¼0.0297), 733 parameters, R1 [I>2
s
(I)]¼0.0421, wR2
ꢁꢁ3
[all data]¼0.1134, largest diff. peak and hole: 0.362 and ꢁ0.195 e A
.
6.4.3. Sphingolipid turnover. Sphingolipids were labeled by in-
cubating B16 cells for 6 h with 0.33
presence or absence of 5 M of compound 3. Then, cells were
m
Ci/mL [3-3H]-sphingosine in the
6.3.13. (3aS,6S,6aS)-6-(4-Fluorophenethyl)tetra-hydrofuro[3,4-d]ox-
azol-2(3H)-one (18). Prepared from N-PMB derivative 17 (56.0 mg,
0.15 mmol) according to the general A. The crude material was pu-
rified by column chromatography on SiO2 eluted with CH2Cl2/EtOAc
m
washed twice with PBS, harvested and centrifuged. Lipids were
extracted and separated by TLC using chloroform/methanol/water
(100:42:6, by vol). Radiolabeled sphingolipids were detected using
a Berthold radiochromatoscanner, identified using lipid standards
and then scraped before counting radioactivity by liquid scintillation.
(60:40 to 50:50) to give 18 (35.2 mg, 93%) as a white solid. ½a D20
þ94.0
ꢂ
(c 1.4, CHCl3); IR (film) nNH 3272, n
C]O 1709 cmꢁ1; 1H NMR (300 MHz,

4262
Y. Salma et al. / Tetrahedron 67 (2011) 4253e4262
ꢀ
ꢀ
7. Genisson, Y.; Lamande, L.; Salma, Y.; Andrieu-Abadie, N.; Andre, C.; Baltas, M.
Tetrahedron: Asymmetry 2007, 18, 857e864.
6.4.4. SMS activity. B16 cells were incubated for 6 h with 5 mM of
Jaspine B or compound 3 in the absence of FCS. Then, 5
mM
8. For a review see: Abraham, E.; Davies, S. G.; Roberts, P. M.; Russell, A. J.;
C6eNBDeceramide was added to the medium as an ethanolic so-
lution. After incubation for 2 hours at 37 ꢀC, cellular lipids were
extracted with chloroform/methanol (2:1, v/v).37 After centrifuga-
tion (1000ꢃg, 10 min), the lower phases were concentrated under
nitrogen and resolved by TLC developed in chloroform/methanol/
30% ammonia/water (70:30:3:2, by vol). Fluorescent lipids were
eluted from the silica and the specific activity of SMS was de-
termined by quantifying the fluorescence of SM at 470 and 530 nm
for the excitation and emission wavelengths, respectively.
Thomson, J. E. Tetrahedron: Asymmetry 2008, 19, 1027e1047.
9. For a review see: Ballereau, S.; Baltas, M.; Genisson, Y. Curr. Org. Chem. 2011, 15,
ꢀ
953e986 For recent syntheses see Ref. 10e18.
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4478e4481.
11. Reddipalli, G.; Venkataiah, M.; Mishra, M. K.; Fadnavis, N. W. Tetrahedron:
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Gaussian 09.38 Vibrational analysis was performed at the same level
of calculation in order to check the nature of the stationary points
found on the ground state potential energy surface (PES), and to
extract the enthalpic and entropic corrections required for the
calculation of Gibbs energies at 298.15 K. From these optimized
structures, the PES was explored along the reaction coordinate
(OeCsp distance) at the same calculation level. The intrinsic re-
action coordinate was followed using the IRC technique imple-
mented in Gaussian 09 for all located transition states. Solvent
effects were included within self-consistent reaction field (SCRF)
calculations using the polarizable continuum model (PCM, water,
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3
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Acknowledgements
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Supplementary data
Supplementary data contains copies of the 13C NMR spectra for
compounds 3, 6e14, 16e19 as well as Fig. S1 and Table S1. Sup-
plementary data associated with this article can be found in online
version at doi:10.1016/j.tet.2011.04.027.
35. Sheldrick, G. M. SHELXL-97, Program for Crystal Structure Refinement; University
€
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