3,4-Disubstituted oxazolidin-2-ones as constrained ceramide analogs with anticancer activities

Article abstract of DOI:10.1016/j.bmc.2011.09.022

Heterocyclic analogs of ceramide as 3-alkanoyl or benzoyl-4-(1-hydroxy-2- enyl)-oxazolidin-2-ones were designed by binding of primary alcohol and amide in sphinogosine backbone as a carbamate. They were synthesized by addition of acyl halide to the common

Full text of DOI:10.1016/j.bmc.2011.09.022

Bioorganic & Medicinal Chemistry 19 (2011) 6174–6181
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
3,4-Disubstituted oxazolidin-2-ones as constrained ceramide analogs
with anticancer activities
Alok Singh a, Hyun-Joon Ha a, , Jungchan Park b, Jun Hee Kim c, Won Koo Lee c,
⇑
⇑
a Department of Chemistry, Hankuk University of Foreign Studies, Yongin 449-791, Republic of Korea
b Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yongin 449-791, Republic of Korea
c Department of Chemistry, Sogang University, Seoul 121-742, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Heterocyclic analogs of ceramide as 3-alkanoyl or benzoyl-4-(1-hydroxy-2-enyl)-oxazolidin-2-ones were
designed by binding of primary alcohol and amide in sphinogosine backbone as a carbamate. They were
synthesized by addition of acyl halide to the common ring 4-(1-t-butyldimethylsilyloxyhexadec-2-enyl)-
oxazolidin-2-one which was elaborated from chiral aziridine-2-carboxylate including stereoselective
reduction and ring opening reactions as key steps. Other analogs with different carbon frame at C4
position which is corresponding to the sphingoid backbone were prepared from 3-cyclopentanecarbon-
yl-4-(1-t-butyldimethylsilyloxybut-2-enyl)-oxazolidin-2-one and straight and cyclic alkenes by cross
metathesis. All compounds were tested as antileukemic drugs against human leukemia HL-60 cells. Many
of them including propionyl, cyclopentanoyl and p-nitrobenzoyl-4-(1-hydroxyhexadec-2-enyl)-oxazoli-
din-2-ones showed better antileukemic activities than natural C2-ceramide with good correlation
between cell death and DNA fragmentation. There is a drastic change of the activities by the carbon chain
lengths at C4 position. Cytotoxicity was induced by caspase activation without significant accumulation
of endogenous ceramide concentration or any perturbation of ceramide metabolism.
Received 9 August 2011
Revised 9 September 2011
Accepted 10 September 2011
Available online 16 September 2011
Keywords:
Ceramide
Oxazolidin-2-one
Sphingoid
Antileukemic
Apoptosis
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
myelin bearing additional phosphocholine to ceramide is also
important pathway for ceramide3 as a secondary messenger by
Sphingolipids including ceramide, sphingomyelin, cerebroside,
and ganglioside are unique membrane components of all eukary-
otic cells.1 They play important roles in the regulation of cell
proliferation, differentiation, migration, survival, and cell-death
via closely related metabolic pathways.1,2 Among all metabolites
ceramide is one of the most important molecules as a secondary
messenger of sphingolipid signaling pathway.2
The main structural components of ceramide (1) are a long alkyl
chain with 2-amino-1,3-diols and C4,5-trans-olefin as the sphingo-
sine backbone and an N-acyl group. In mammalian tissue the most
sphingomyelinase4 in response to extracellular agents and stresses
including chemotherapeutic agents5 tumor necrosis factor
a
a (TNF-
),6 and ionizing radiation.7 Thedynamic balance of the sphingolipid
metabolites is an importantfactor whether a cell undergoes prolifer-
ation or programmed death so called apoptosis.8 The close relation-
ship is found between ceramide and its hydrolytic products
sphingosine triggered by growth factors such as platelet-driven
growthfactors. Ceramideasanimportantstimulatingfactorofapop-
tosis is hydrolyzed by ceramidase to sphingosine followed by activa-
tion of kinase to yield sphingosine-1-phosphate with cell growth.
Lots of sphingosine analogs were isolated from natural sources
and synthesized based on the structural modification of sphingoid
backbones, and their biological activities were evaluated. Among
them the synthetic analog safingol and the marine natural product
spisulosin were good examples. Safingol is an inhibitor of protein
kinase C9 and spisulosin inhibits the activity of Rho protein.10
Few years ago we envisaged the synthetic method for the construc-
tion of sphingoid backbone and their analogs including anticancer
agent’s spisulosin and safingol based on the stereospecific reduc-
tion of 2-acylaziridine and aziridine ring opening reactions.11
Among the enormous increase in the research of sphingolipid
metabolism the central metabolite ceramide attracts most atten-
tions due to its important role at the stages of apoptosis with a
common sphingoid base is D-erythro-C18-sphingosine [(2S,3R,4E)-
2-amino-octadec-4-en-1,3-diol, (E)-sphing-4-enine] and the typical
acyl group attached on the amine has the chain length of 16–24. The
structural changes of ceramide at the primary alcohol occur at the
cellular metabolic system by addition of phosphate, phosphocholine
and carbohydrates to form ceramide-1-phosphate (2), sphingomye-
lin (3) and glucosylceramide (4) (Fig. 1). Specifically ceramide is gen-
erated from sphingosine by ceramide synthase with an additional
fatty acid chain to the sphingoid backbone. Hydrolysis of sphingo-
⇑
Corresponding authors. Tel.: +82 31 3304369; fax: +82 31 3304566 (H.-J.H.);
tel.: +82 1 705 8449 (W.K.L.).
E-mail addresses: hjha@hufs.ac.kr (H.-J. Ha), wonkoo@sogang.ac.kr (W.K. Lee).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.022

A. Singh et al. / Bioorg. Med. Chem. 19 (2011) 6174–6181
6175
O
This analog retains the same conformation with the allylic alco-
hol system as for the natural substrate that is one of the essential
structural elements to keep the biological activity with the gener-
ation of reactive oxygen species.2c,17b Furthermore, in this analog
the free primary hydroxyl group of the natural substrate was
bound to the oxazolidin-2-one ring that may block metabolic
perturbation occurring at the hydroxyl group of C-1 of the sphing-
oid backbone. Thereby we decided to prepare 3-alkanoyl or
benzoyl-4-(1-hydroxy-2-enyl)-oxazolidin-2-ones as heterocyclic
analogs of ceramide and to test for antileukemic drugs against hu-
man leukemia HL-60 cells.
O
HN
C15H31
HN
C15H31
(
)
R
HO
C13H27
(
)
R
RO
C13H27
(S)
(
)
S
OH
Ceramide (1)
OH
2 R = P(=O)(O-)2
R = P(=O)(O )(OCH2CH2N Me3)
R = Glucose
-
+
3
4
Figure 1. Structures of ceramide (1) ceramide-1-phospahate (2), sphingomyelin (3)
and glucosylceramide (4).
selective toxicity to malignant but not to normal cells.12 Various
analytical methods show that ceramide is accumulated during
the initiation stage of apoptosis.3 The central molecule in the
sphingolipid metabolism, ceramide, is known to induce apoptosis,
senescence,13 and growth arrest14 in many human cancers. There-
fore, many researchers have focused on designing ceramide
analogs with the same apoptotic activity as ceramide. They lead
synthesizing dozens of active molecules reported recently, many
of which have greater inhibitory effect on the enzymes involved
in ceramide metabolism.15
2. Results and discussion
All of the designed compounds were synthesized from commer-
cially available (2S)-aziridine-2-carboxylate (6) shown in the
following Scheme 1.
The aziridine carboxylate
6 was reacted with N,O-dim-
ethylhydroxylamine in the presence of i-PrMgBr at 0 °C to form
the Weinreb’s amide 7 in 92% yield followed by addition of 1-pen-
tadecynyl lithium to yield 8 in quantitative yield. Acyl aziridine 8
was selectively reduced to erythro-amino alcohol 9 to have the
right stereochemistry of ceramide with NaBH4 and ZnCl2 via the
chelation-controlled transition state in 98% yield.11 Then alkyne 9
Exogenous ceramide actually induces apoptotic programmed
cell death. In particular, cell-permeable ceramide with short acyl
group such as C2-Cer (N-acetyl-D-erythro-C18-sphingosine) and
C8-Cer (N-octyl-
D
-erythro-C18-sphingosine) inhibit tumor cell
was reduced by LAH to yield N-[(R)-a-methylbenzyl]aziridin-
growth and induce apoptosis in leukemia cell lines.16 The
ceramides with longer alkyl chain than C8-Cer including natural
ceramide (C18-Cer) are too lipophilic to pass through the cell
2(S)-yl-hydroxy-1-pentadecene in 86% yield whose hydroxy group
was protected by TBDMS in 93% yield. The aziridine ring was regio-
selectively opened by the reaction with acetic acid in CH2Cl2 to
give acyclic compound 10 in 73%. The O-acetyl group in 10 was
hydrolyzed by KOH in ethanol to yield the free alcohol at which
stage oxazoldin-2-one ring was introduced by the reaction with
membrane. Dihydroceramide (DHCer) with saturated
D-erythro-
C18-sphinganine does not have any biological activity which
seems to be due to the lack of essential trans allylic alcohol.17 For
this reason, many synthetic and natural ceramide analogs were
prepared and evaluated as anticancer agents by altering the back-
bone and by changing two hydroxy and one amine functional
groups. Those include ceramide analogs whose backbone was
modified with various aromatic or heteroaromatic systems instead
of simple long alkyl chain of sphingoid base with free hydroxyl
group at the end of the backbone.18 However, most of them were
less active than C2-Cer. Furthermore, acyclic ceramide analogs
bearing primary alcohol in sphingoid backbone perturb or at least
have a chance to perturb the metabolic pathways by its possible
involvement in various biological reactions.
Constrained ceramide analogs without the primary alcohol may
overcome this drawback of metabolic perturbation. A good exam-
ple was shown by uracil ring compound in which the polar portion
of ceramide was replaced.19 This compound has very potent
growth-inhibitory effect of CCRF-CEM human leukemia cells to
trigger apoptosis by the occurrence of DNA and nuclear fragmenta-
tion.19 However, these uracil analogs do not bear an allylic alcohol
system and do have aromaticity in the main ring part, which are
quite different from the real conformational alignment of acyclic
C2-Cer.
1,1’-carbodiimidazole in 70% yield for two steps. The a-methylben-
zyl group attached on the nitrogen was removed by Na in liq. NH3
at À78 °C to give the backbone ring 11 in 77% yield. The parent ring
(11) was readily acylated on the nitrogen using acid chloride in the
presence of a suitable base. Sodium hexamethyldisilazane was the
most effective base being applicable to various acyl chlorides. The
target molecules (5a–5m) were prepared from the parent ring
compound 11 by the same reaction sequence with the correspond-
ing acid chlorides followed by desilylations in over 85–95% yield in
all cases over two steps.
All of these compounds were evaluated as potential antileuke-
mic agents against human leukemia HL-60 cells with C2-Cer and
C2-DHCer as positive and negative references, respectively by
MTT assay whose results were shown in Figure 3.22 Human leuke-
mia HL-60 cells were treated with 20 lM of each constrained
ceramide analogs with references for 16 h. All tested compounds
(5a–5m) showed activities with drastic difference by the N-acyl
Recently we reported a short communication for the synthesis
and biological activities of new constrained ceramide analogs
which are closer to the real conformational and electronic proper-
ties of ceramide on the basis of oxazolidin-2-one ring (5).20 Analogs
with oxazolidin-2-one ring were designed to combine the primary
alcohol at C1 and the amide nitrogen at C2 of ceramide together by
additional carbonyl group. This conformation was deduced by
early observation that there are strong hydrogen-bonding among
alcoholic hydrogens and the amide nitrogen.21 An analog, N-hexa-
decanoyl compound 5c, mimicked naturally occurring acyclic
C16-Cer whose conformational similarity was evaluated by the
comparison with the minimum energy conformers calculated by
density functional model.20 The overlay structure of those two
compounds, C16-Cer and 5c, shows similarity in Figure 2.
O
O
O
N
C15H31
C13H27
HN
C15H31
C13H27
O
HO
OH
OH
5c
1
Figure 2. Overlay of naturally occurring C16-ceramide (1) and its oxazolidin-2-one
analog (5c). Calculation was carried out with the commercial product SpartanÒ 04.
The overlay structures were deduced by locking the bonds associated with nitrogen
for comparison.

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A. Singh et al. / Bioorg. Med. Chem. 19 (2011) 6174–6181
Me
Me
Me
O
O
O
ii
i
Ph
N
Ph
N
Ph
N
OMe
OEt
N
C
12H25
H
H
H
Me
6
7
8
iii
Ph
Me
O
OH
v
HN Me
iv
Ph
N
NH
AcO
O
C12H25
C12H25
C12H25
H
H
OTBS
OTBS
11
10
9
vi
R1 = 5a Methyl
5b Ethyl
(94%)
(92%)
(91%)
(88%)
(85%)
(90%)
(91%)
(95%)
(92%)
(88%)
(86%)
(90%)
(93%)
5h Cinnamyl
5i Phenyl
5j o-Methoxyphenyl
5k p-Nitrophenyl
5c Pentadecyl
5d i-Propyl
5e t-Butyl
5f Cyclopentyl
5g Benzyl
O
O
R1
N
H
5l
1-Naphthyl
O
5m Furyl
C12H25
OH
5
Scheme 1. Reagents and conditions: (i) NH(OMe)CH3, i-PrMgBr, THF, 0 °C (92%); (ii) HCCHCH2C12H25, n-BuLi, À78 °C then 0 °C, 2 h (99%); (iii) ZnCl2, NaBH4, MeOH, À78 °C,
then À15 °C, 1 h (98%); (iv) (a) LAH, 0–60 °C, 8 h (86%); (b) DMAP, t-BuMe2SiCl, rt (93%); (c) AcOH, CH2Cl2, rt, (73%); (v) (a) KOH, EtOH, rt, 1 h (81%); (b) CDI, DBU, rt, 24 h
(87%); (c) Na, NH3, À78 °C (77%); (vi) (a) R1COCl, NaHMDS, 0 °C; (b) Bu4NF, THF, 0 °C.
Figure 3. Relative cell survival of HL-60 cells after treatment with 20
lM of C2-Cer, C2-DHCer and oxazolidin-2-ones (5a–5m) and (14, 15a–15g) for 16 h.
substituents of the oxazolin-2-one ring. N-acetyl compound 5a
mimicking C2-Cer has much lower activity than C2-Cer itself while
a great deal of improvement was observed in the compound 5b
with additional one carbon as ethyl group in R1 of the compound
5. Compound 5c with a long alkyl chain did not show any activity
due to low cell permeability to produce its antileukemic activity
that was also observed in natural C16-Cer.16 The longer carbon
chain than ethyl (5b) such as i-propyl (5d) or butyl (5e) did not
improve the activity much. The cyclopropanyl ring at R1 in the
compound 5f improved the activity drastically which showed the
best activity among all tested compounds with alkyl substituents.
Compounds with benzyl (5g) and cinnamyl (5h) were not active.
Some of the compounds possessing benzoyl (5i–5k) rather than
alkanoyl group (5a–5h) showed good activities. Their activities
were dependent to the substituents of phenyl ring. Especially the
compound (5k) with p-nitrophenyl for R1 showed very potent
activity. Aryl substituents other than phenyl for R1 group in the
compounds 5 did not improve the activity among the tested com-
pounds including naphthyl (5l) and furyl (5m) substituents. The
origin of the activity difference among all tested compounds is
speculated not from the simply physical properties such as
lipophilicity or polarity but from the unique structural features
of each compound.
We decided to find out whether the cytotoxicity induced by
these analogs or the reduction of HL60 cells was caused by the
apoptosis. The characteristics of cell death were verified by the
analysis of DNA-fragmentation induced by these molecules. DNA-
laddering of fragments was observed after 15 h of incubation of
HL-60 cells with 20 lM of C2-Cer, C2-DHCer and oxazolidin-2-ones
(5a, 5b, 5c, 5d, 5f, 5i and 5k) shown in Figure 4. The picture showed
the clear co-relationship between anticancer activity and DNA-
fragmentation.
The active compounds such as 5b (R1 = ethyl) and 5i (R1 = phe-
nyl) showed similar or a little more amount of DNA fragmentation
as white bands than for C2-Cer while inactive 5a (R1 = methyl) and
5c (R1 = pentadecyl) had similar band to C2-DHCer’s on the picture.
All active compounds 5d (R1 = i-propyl), 5f (R1 = cyclopentyl), 5i
(R1 = phenyl) and 5k (p-nitrophenyl) also showed DNA-fragmented
bands. This demonstrates cell death was caused by the apoptotic
process. A good correlation between cell death of HL-60 cells and
DNA fragmentation is solid evidence that the cytotoxicity of the
constrained ceramide analogs is originated from apoptosis.

A. Singh et al. / Bioorg. Med. Chem. 19 (2011) 6174–6181
6177
chain length less than 12 in R2. Only 15d consisted of 15 carbons at
the backbone, which is shorter than natural ceramide by three
carbon units, shows comparably good activity. The compounds
with cyclopentyl, cyclohexyl and benzyl at the backbone site
showed no activity either. This indicates that the hydrophobic
binding site of the receptor inducing anticancer activity is quite
deep enough to accommodate carbon chains with certain length
and is narrow enough not to get the cyclic compounds. This is
the first detailed study on the structure and activity relationship
in the pro-apoptotic process with diverse oxazolidin-2-ones as
constrained ceramide analogs.
3. Conclusion
Figure 4. DNA fragmentation of HL-60 cells by the treatment with 20 lM of C2-Cer,
C2-DHCer and some representative oxazolidin-2-ones (5a, 5b, 5c, 5d, 5f, 5i, 5k).
Heterocyclic analogs of ceramide as 3-alkanoyl or benzoyl-4-(1-
hydroxy-2-enyl)-oxazolidin-2-ones were designed by binding of
primary alcohol and amide in sphingosine backbone as a carba-
mate. These were based on the unique butterfly conformer of
natural ceramide folded by the strong internal hydrogen bonding
between amide and two alcohols at C1 and C3 and by the hydro-
phobic interaction between two long alkyl chain of sphingosine
backbone and fatty acid. Some of them with different acyl group
at the ring nitrogen were synthesized by addition of acyl halide
to the common ring 4-(1-t-butyldimethylsilyloxyhexadec-2-
enyl)-oxazolidin-2-one which was elaborated from chiral aziri-
dine-2-carboxylate including stereoselective reduction and ring
opening reactions. Other analogs with different carbon backbone
at C4 were prepared from 3-cyclopentanecarbonyl-4-(1-t-butyl-
dimethylsilyloxybut-2-enyl)-oxazolidin-2-one by cross metathe-
sis. All compounds were tested as antileukemic drugs against
human leukemia HL-60 cells. Many of them including propionyl,
cyclopentanoyl and p-nitrobenzoyl-4-(1-hydroxyhexadec-2-enyl)-
oxazolidin-2-ones showed better antileukemic activities than
natural C2-Cer with good correlation between cell death and
DNA fragmentation. There is a drastic change of the activities by
the carbon chain lengths at C4 position which is corresponding
to the sphingoid backbone. Almost no activity was observed from
the compounds bearing the chain length less than twelve of
sphingoid backbone. Cytotoxicity was induced by caspase activa-
tion without significant accumulation of endogenous ceramide
concentration or any perturbation of ceramide metabolism.
The cellular concentration of ceramide, measured three times
by the reported method23 after the treatment of the active analogs,
showed minor changes by less than 15%, which means the oxazoli-
din-2-one ceramide analogs did not inhibit the ceramide metabolic
enzymes to accumulate the cellular concentration of ceramide.
Instead these molecules induced apoptosis through caspase cas-
cade with induction of nuclear factor
jB (NF-jB) DNA binding
and caspase-3 activation as exogeneous C-2 and C-6-ceramides
do.16b
All of the compounds synthesized and tested up to this point
have 1-hydroxyhexadec-2-ene at C4 of oxazolidin-2-one24 which
is the same backbone with eighteen carbons as the sphingoid’s.
For the investigation of the effect by changing the sphingoid back-
bone were designed and synthesized many analogs bearing differ-
ent carbon numbers with and without ring. They have different
sphingoid backbones consisted of different carbon chains at R2 in
Scheme 2 bearing the olefin at the right position that the natural
sphingoid has. The cyclopentanoyl group was attached at the ring
nitrogen because it showed the best biological activity among
other substituent. Those included compounds having four (15a),
six (15b), eight (15c) and ten (15d) straight carbons for the R2
group. Others are cyclopentyl (15e), cyclohexyl (15f) and benzyl
(15g) groups. All of these were synthesized from the methathesis25
between the common intermediate 14 and the corresponding
olefins including 1-hexene, 1-octene, 1-decene, 1-dodecene, vinyl-
cyclopentane, vinylcyclohexane and allylbenzene in the presence
of the Grubbs’s 2nd generation catalyst in high yields (Scheme 2).
All of them except 15d showed the worse activities compared to
the C2-Cer. There is a drastic change of the activities by the carbon
chain length corresponding to the sphingoid backbone. Almost no
activity was observed from the compounds (15a–15c) bearing the
4. Experimental
All non-aqueous reactions were run in flame-dried glassware
under a positive pressure of nitrogen with exclusion of moisture
from reagents and glassware using standard techniques for
O
O
Me
O
O
O
i
ii
Ref-25
NH
N
Ph
N
OEt
O
Me
Me
88%
99%
H
H
H
OTBS
O
OTBS
13
12
6
R2=
15a -CH2(CH2)2CH3 (88%)
O
15b
15c
- CH2(CH2)4CH3 (86%)
(88%)
O
O
O
- CH2(CH2)6CH3
N
H
iii
N
15d - CH2(CH2)8CH3 (89%)
O
R2
Me
15e
15f
- Cyclopentyl
- Cyclohexyl
(78%)
(75%)
(82%)
H
OH
OH
15g - Benzyl
14
15
Scheme 2. Reagents and conditions: (i) Sodium bis(trimethylsilyl)amide, cyclopentanecarbonyl chloride, THF, À78 °C, 2 h; (ii) TBAF, THF, 0 °C, 1 h, then at rt, 2 h; (iii)
benzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(tricyclohexylphosphine)ruthenium, CH2CH2, 24 h.

6178
A. Singh et al. / Bioorg. Med. Chem. 19 (2011) 6174–6181
manipulating air-sensitive compounds. Anhydrous solvents were
obtained using standard drying techniques. Unless stated other-
wise and commercial grade reagents were used without further
MgSO4, filtered, and concentrated in vacuo. Purification by silica
flash chromatography gave product in 93% yield.11 To a solution
of substrate obtained above reaction in CH2Cl2 under a nitrogen
atmosphere at room temperature was added AcOH. The mixture
was stirred for 10 h and then quenched with 10 mL of saturated
NaHCO3 solution. The organic layer was separated and the aqueous
layer was extracted with CH2Cl2. The combined organic extracts
were washed with brine, dried over anhydrous MgSO4, filtered,
and concentrated in vacuo. Purification by column chromatogra-
phy gave 10 in 73% yield: 1H NMR (500 MHz, CDCl3) d 7.29–7.22
(m, 5H), 5.62 (dt, J = 15.4 Hz, 1H), 5.30 (dd, J = 14.7 Hz, 1H), 4.16–
4.11 (m, 3H), 3.91 (q, J = 6.6 Hz, 1H), 2.52–2.49 (m, 1H), 2.10–
1.98 (m, 5H), 1.29–1.19 (m, 25H), 0.85 (s, 12H), 0.00 (s, 3H),
À0.02 (s, 3H); HRMS (ESI) calcd for [C34H61NO3Si+H]+: 560.4499,
found 560.4504.
purification. (2S)-1-[(R)-a-methylbenzyl]-2-aziridinecarboxylate
ethyl esters were purchased from Imagene. Their menthol esters
were also purchased from Aldrich. Reactions were monitored by
analytical thin-layer chromatography (TLC) performed on pre-
coated, glass-backed silica gel plates. Visualization of the devel-
oped chromatogram was performed by UV absorbance, ninhydrin,
phosphomolybdic acid or iodine. Flash chromatography was per-
formed on 230–400 mesh silica gel with the indicated solvent sys-
tems. Melting points are uncorrected. Routine nuclear magnetic
resonance spectra were recorded either on Varian Gemini 200
(200 MHz) or Varian Gemini 400 (400 MHz) spectrometers. Chem-
ical shifts for 1H NMR spectra are recorded in parts per million
from tetramethylsilane with the solvent resonance as the internal
standard (CDCl3, d 7.27 ppm and CD3OD d 3.31 ppm). Data are
reported as follows: chemical shift, multiplicity (s = singlet,
d = doublet, t = triplet, q = quartet, qn = quintet, m = multiplet,
and br = broad), coupling constant in Hertz, and integration. Chem-
ical shifts for 13C NMR spectra are recorded in parts per million
from tetramethylsilane using the central peak of the solvent reso-
nance as the internal standard (CDCl3, d 77.00 ppm and CD3OD d
49.15). All spectra were obtained with complete proton
decoupling.
4.4. (S)-4-[(R,E)-1-t-Butyldimethylsilyloxyhexadec-2-
enyl]oxazolidin-2-one (11)
To a solution of 10 in EtOH at room temperature was added
KOH. The mixture was stirred for 1 h at room temperature and
then quenched with 10 mL of water. The product was extracted
with CH2Cl2 and the combined organic extracts were washed with
brine, dried over MgSO4, filtered, and concentrated. Purification by
column chromatography gave product in 81% yield. Then this was
dissolved in CH2Cl2. The resultant solution was stirred followed by
adding DBU and carbonyl diimidazole at room temperature. The
mixture was stirred for overnight and then concentrated in vacuo.
Purification by silica gel flash chromatography gave product in 87%
yield. Anhydrous NH3 was condensed into a flask containing a lith-
ium metal maintained at À78 °C until a dark blue color persisted
and then added tert-butyl alcohol. The mixture was stirred for
5 min at À78 °C and then added substrate in THF. The resulting
mixture was stirred for 30 min and then quenched with water.
The mixture was extracted with EtOAc. The combined extracts
were washed with brine, dried over MgSO4, filtered, and concen-
trated under reduced pressure. Purification by silica gel flash chro-
matography gives product 11 in 77% yield: 1H NMR (500 MHz,
CDCl3) d 5.64 (dt, J = 15, 5 Hz, 1H), 5.59 (s, 1H), 5.38 (dd J = 15,
5 Hz, 1H), 4.32 (dd, J = 19, 9 Hz, 1H), 4.29–4.27 (m, 1H), 4.00 (t,
J = 6 Hz, 1H), 3.89–3.84 (m, 1H), 2.06–2.01 (m, 2H), 1.41–1.38 (m,
22H), 0.92 (s, 12H), 0.07 (s, 3H), 0.04 (s, 3H); HRMS (ESI) calcd
for [C25H49NO3Si+H]+: 440.3560, found 440.3569.
4.1. 1-{(S)-1-[(S)-1-phenylethyl]aziridin-2-yl}hexadec-2-yn-1-
one (8)11
To a solution of pentadecyne (1.5 equiv) in anhydrous THF
(0.3 M) under nitrogen at À78 °C was added n-BuLi (1.3 equiv).
The mixture was stirred for 30 min, and then treated with 7 (1 g,
4.27 mmol) via canula at À78 °C. The mixture was stirred for 1 h
at À78 °C and was warmed to room temperature and then treated
with 10 mL of water. The organic layer was separated and the
aqueous layer was extracted with EtOAc (20 mL Â 4). The com-
bined organic extracts were dried over anhydrous MgSO4, filtered,
and concentrated in vacuo. Purification by silica gel flash chroma-
tography gave 1.45 g of product 8 in 99% yield.
4.2. (R)-1-{(S)-1-[(R)-1-phenylethyl]aziridin-2-yl}hexadec-2-yn-
1-ol (9)11
To the solution of 8 (1 g, 2.62 mmol) in MeOH (0.3 M) at À78 °C
was added ZnCl2 (1.2 equiv). The solution was stirred for 30 min,
and NaBH4 (2 equiv) was added at À78 °C. The mixture was stirred
for 30 min, and 20 mL of water was added. The organic layer was
separated. The aqueous layer was extracted with CH2Cl2, and the
combined organic extracted were dried, filtered, and concentrated
in vacuo. Purification by silica gel flash chromatography provided 9
in 98% yield.
4.5. General procedure for acylation and desilylation procedure
(5)
To a stirred solution of 11 in anhydrous THF was added sodium
bis(trimethylsilyl)amide (1.2 equiv) at À78 °C. After 10 min at
À78 °C, the mixture was added acylchloride (1.3 equiv) at À78 °C.
The reaction was complete according to TLC, extracted with
EtOAc/H2O, dried over MgSO4, and purified by column chromatog-
raphy to give the coupled product. A solution of substrate in anhy-
drous THF was added TBAF at 0 °C. After 4 h, the reaction mixture
was complete, extracted with EtOAc/H2O, dried over MgSO4, and
purified by column chromatography to give final product (5) in
85–95% yield after two steps.
4.3. (E,2S,3R)-2-[(R)-1-Phenylethylamino]-3-(t-
butyldimethylsilyloxy)-octadec-4-enyl acetate (10)
To a stirred solution of substrate in anhydrous THF was added
LAH at room temperature. After 3 h, the reaction was completed
according to TLC, quenched by aqueous KHSO4, extracted with
EtOAc/H2O, dried over MgSO4, and purified by column chromatog-
raphy to give the reaction product in 86% yield. The stirred solution
of the reduced substrate in CH2Cl2 was added DMAP at room tem-
perature. The mixture was added TBDMSCl at room temperature.
The reaction mixture was stirred for 17 h at room temperature
and was treated with 10 mL of saturated NaHCO3 solution. The
organic extracts were washed with brine, dried over anhydrous
4.5.1. (S)-3-Acetyl-4-[(R,E)-1-hydroxyhexadec-2-
enyl]oxazolidin-2-one (5a)
Yield: 91%; 1H NMR (300 MHz, CDCl3) d 5.86 (dt, J = 11.2, 4 Hz,
1H), 5.39–5.25 (m, 2H), 4.45 (t, J = 9 Hz, 1H), 4.30–4.28 (m, 1H),
4.04–4.01 (m, 1H), 2.06 (q, J = 5 Hz, 2H), 1.48–1.39 (m, 23H), 1.12
(t, J = 7 Hz, 3H), 0.88 (t, J = 5 Hz, 3H); HRMS (ESI) calcd for
[C21H37NO4+H]+: 368.2801, found 368.2814.

A. Singh et al. / Bioorg. Med. Chem. 19 (2011) 6174–6181
6179
4.5.2. (S)-4-[(R,E)-1-Hydroxyhexadec-2-enyl]-3-
propionyloxazolidin-2-one (5b)
1H), 2.05 (d, J = 7.5 Hz, 1H), 1.25 (br s, 22H), 0.88 (t, J = 6.6 Hz,
3H); 13C NMR (75 MHz, CDCl3) d 170.1, 146.4, 139.8, 129.2,
128.5, 122.3, 117.4, 75.0, 66.5, 54.9, 32.7, 32.2, 30.0, 29.9, 29.6,
29.4, 29.0, 22.9, 14.4; HRMS (ESI) calcd for [C28H41NO4+H]+:
456.3114, found 456.3118.
Yield: 95%; 1H NMR (300 MHz, CDCl3) d 5.88 (dt, J = 11, 4 Hz,
1H), 5.23–5.32 (m, 2H), 4.47 (t, J = 9 Hz, 1H), 4.31–4.29 (m, 1H),
4.04–4.01 (m, 1H), 2.35 (q, J = 5 Hz, 2H), 2.08 (q, J = 5 Hz, 2H),
1.41–1.36 (m, 23H), 1.14 (t, J = 7 Hz, 3H), 0.89 (t, J = 5 Hz, 3H);
HRMS (ESI) calcd for [C22H39NO4+H]+: 382.2957, found 382.2951.
4.5.9. (S)-3-Phenyl-4-[(R,E)-1-hydroxyhexadec-2-
enyl]oxazolidin-2-one (5i)
4.5.3. (S)-4-[(R,E)-1-Hydroxyhexadec-2-enyl]-3-
palmityryloxazolidin-2-one (5c)
Yield: 92%; mp = 82 °C; 1H NMR (300 MHz, CDCl3) d 8.04 (d,
J = 8 Hz, 2H), 7.59 (t, J = 8 Hz, 1H), 7.46 (t, J = 8 Hz, 2H), 5.96 (m,
2H), 5.55 (m, 2H), 4.49 (t, J = 8 Hz, 1H), 4.38 (m, 1H), 4.05 (m,
1H), 2.08 (q, J = 7 Hz, 2H), 1.24 (m, 22H), 0.89 (t, J = 7 Hz, 3H);
HRMS (ESI) calcd for [C26H39NO4+H]+: 430.2957, found 430.2954.
Yield: 92%; 1H NMR (300 MHz, CDCl3) 5.88 (dt, J = 12, 4 Hz, 1H),
5.19–5.33 (m, 3H), 4.44 (t, J = 9 Hz, 1H), 4.26 (m, 1H), 4.01 (m, 1H),
2.34 (t, J = 7 Hz, 2H), 2.07 (q, J = 7 Hz, 2H), 1.70–1.68 (m, 3H), 1.28–
1.18 (m, 48H), 0.89 (t, J = 7 Hz, 3H); HRMS (ESI) calcd for
[C35H65NO4+H]+: 564.4992, found 564.4983.
4.5.10. (S)-4-[(R,E)-1-Hydroxyhexadec-2-enyl]-3-(2-
tolyl)oxazolidin-2-one (5j)
4.5.4. (S)-4-[(R,E)-1-Hydroxyhexadec-2-enyl]-3-
(isobutyryl)oxazolidin-2-one (5d)
Yield: 91%; 1H NMR (300 MHz, CDCl3) d 7.88 (dd, J = 4.8, 1.8 Hz,
1H), 7.52 (td, J = 9.0, 1.8 Hz, 1H), 7.1–6.9 (m, 2H), 6.1–5.9 (m, 2H),
5.6–5.4 (m, 3H), 4.51 (t, J = 9.0 Hz, 1H), 4.37 (dd, J = 9.0, 4.8 Hz, 1H),
4.2–4.1 (m, 1H), 3.94 (s, 3H), 2.12 (d, J = 6.6 Hz, 1H), 2.07 (d,
J = 7.2 Hz, 1H), 1.4–1.2 (m, 21H), 0.88 (t, J = 6.6 Hz, 3H); 13C NMR
(75 MHz, CDCl3) d 165.4, 159.2, 158.9, 151.3, 139.3, 133.9, 131.7,
121.9, 120.3, 112.0, 74.8, 66.1, 55.9, 54.6, 32.4, 31.9, 29.6, 29.5,
29.4, 29.3, 29.2, 29.1, 29.7, 22.7, 14.1; HRMS (ESI) calcd for
[C27H41NO5+H]+: 460.3063, found 460.3070.
Yield: 88%; 1H NMR (300 MHz, CDCl3) d 5.88 (dt, J = 8.4, 6.6 Hz,
1H), 5.4–5.25 (m, 2H), 4.43 (t, J = 9.0 Hz, 1H), 4.26 (dd, J = 9.0,
5.1 Hz, 1H), 4.03–3.97 (m, 1H), 2.63–2.53 (m, 1H), 2.08 (d,
J = 6.0 Hz, 1H), 2.03 (d, J = 6.6 Hz, 2H), 1.4–1.2 (m, 22H), 1.19 (d,
J = 3.3 Hz, 3H), 1.17 (d, J = 3.3 Hz, 3H), 0.88 (t, J = 6.6 Hz, 3H); 13C
NMR (75 MHz, CDCl3) d 176.0, 159.1, 138.7, 122.2, 73.9, 65.9,
54.6, 34.1, 32.3, 31.9, 29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 28.7, 22.6,
18.9, 18.7, 14.0; HRMS (ESI) calcd for [C23H41NO4+H]+: 396.3114,
found 396.3121.
4.5.11. (S)-4-[(R,E)-1-Hydroxyhexadec-2-enyl]-3-(4-
nitrophenyl)-oxazolidin-2-one (5k)
Yield: 88%; 1H NMR (300 MHz, CDCl3) d 8.28 (dd, J = 6.9, 1.8 Hz,
2H), 8.20 (dd, J = 6.9, 1.8 Hz, 2H), 6.1–5.9 (m, 2H), 5.6–5.4 (m, 2H),
4.53 (t, J = 9.0 Hz, 1H), 4.37 (dd, J = 9.0, 4.5 Hz, 1H), 4.2–4.1 (m, 1H),
4.5.5. (S)-4-[(R,E)-1-Hydroxyhexadec-2-enyl]-3-
(pivaloyl)oxazolidin-2-one (5e)
Yield: 86%; 1H NMR (300 MHz, CDCl3) d 5.91–5.81 (m, 1H), 5.44
(br s, 1H), 5.37–5.27 (m, 2H), 4.42 (t, J = 9.0 Hz, 1H), 4.27 (dd, J = 9.0,
5.1 Hz, 1H), 4.03–4.96 (m, 1H), 2.08 (d, J = 6.6 Hz, 1H), 2.03 (d,
J = 4.5 Hz, 2H), 1.36–1.22 (m, 22H), 1.21 (s, 9H), 0.88 (t, J = 6.6 Hz,
3H); 13C NMR (75 MHz, CDCl3) d 169.8, 138.4, 122.2, 73.7, 70.5,
65.8, 54.7, 32.3, 31.9, 29.6, 29.4, 29.0, 28.7, 27.0, 22.6, 14.1; HRMS
(ESI) calcd for [C24H43NO4+H]+: 410.3270, found 410.3268.
2.09 (q, J = 6.9 Hz, 2H), 1.4–1.2 (m, 22H), 0.88 (t, J = 6.9 Hz, 3H); 13
C
NMR (75 MHz, CDCl3) d 163.6, 159.2, 140.4, 134.8, 130.8, 123.7,
121.3, 99.9, 76.4, 65.9, 54.6, 32.4, 31.9, 29.6, 29.5, 29.4, 29.3,
29.2, 29.1, 28.6, 22.6, 14.1; HRMS (ESI) calcd for [C26H38N2O6+H]+:
475.2808, found 475.2815.
4.5.12. (S)-4-[(R,E)-1-Hydroxyhexadec-2-enyl]-3-(1-naphthoyl)-
oxazolidin-2-one (5l)
4.5.6. (S)-4-[(R,E)-1-Hydroxyhexadec-2-enyl]-3-
(cyclopentyl)oxazolidin-2-one (5f)
Yield: 85%; 1H NMR (300 MHz, CDCl3) d 8.89 (d, J = 7.2 Hz, 1H),
8.20 (d, J = 7.2 Hz, 1H), 8.07 (d, J = 7.1 Hz, 1H), 7.92 (d, J = 7.1 Hz,
1H), 7.68–7.51 (m, 4H), 6.06 (dt, J = 15.0, 6.6 Hz, 1H), 5.76 (br s,
1H), 5.64–5.50 (m, 2H), 4.55 (t, J = 9.0 Hz, 1H), 4.40 (dd, J = 9.0,
4.8 Hz, 1H), 4.25–4.19 (m, 1H), 2.12 (dd, J = 13.8, 6.9 Hz, 2H),
1.45–1.25 (m, 21H), 0.88 (t, J = 6.6 Hz, 3H); 13C NMR (75 MHz,
CDCl3) d 172.5, 166.5, 159.4, 139.9, 134.1, 131.7, 130.6, 128.9,
128.3, 126.6, 125.8, 124.8, 122.4, 75.5, 66.5, 55.1, 32.7, 32.2, 29.9,
29.8, 29.7, 29.6, 29.5, 29.4, 29.0, 22.9, 14.4; HRMS (ESI) calcd for
[C30H41NO4+H]+: 480.3114, found 480.3120.
Yield: 90%; 1H NMR (300 MHz, CDCl3) d 5.9–5.8 (m, 1H), 5.44 (br
s, 1H), 5.4–5.2 (m, 2H), 4.45 (t, J = 9.0 Hz, 1H), 4.28 (dd, J = 9.0,
5.1 Hz, 1H), 4.1–3.9 (m, 1H), 2.8–2.7 (m, 1H), 2.11 (d, J = 6.3 Hz,
1H), 2.06 (d, J = 7.5 Hz, 1H), 2.0–1.6 (m, 8H), 1.28 (br s, 22H),
0.88 (t, J = 6.6 Hz, 3H); 13C NMR (75 MHz, CDCl3) d 175.7, 159.0,
138.7, 122.3, 73.9, 65.9, 54.7, 43.9, 32.3, 31.9, 29.6, 29.5, 29.4,
29.3, 29.2, 29.1, 29.7, 25.8, 25.7, 22.6, 14.1; HRMS (ESI) calcd for
[C25H43NO4+H]+: 422.3270, found 422.3277.
4.5.7. (S)-4-[(R,E)-1-Hydroxyhexadec-2-enyl]-3-(2-
phenylacetyl)oxazolidin-2-one (5g)
4.5.13. (S)-3-(Furan-2-carbonyl)-4-((R,E)-1-hydroxyhexadec-2-
enyl)oxazolidin-2-one (5m)
Yield: 93%; 1H NMR (300 MHz, CDCl3) d 7.4–7.2 (m, 5H), 5.86–
5.76 (m, 1H), 5.34–5.22 (m, 2H), 5.09 (br s, 1H), 4.35 (t,
J = 9.0 Hz, 1H), 4.12 (dd, J = 9.0, 5.1 Hz, 1H), 3.98–3.92 (m, 1H),
3.65 (s, 2H), 2.02 (q, J = 6.9 Hz, 2H), 1.35–1.24 (m, 22H), 0.88 (t,
J = 6.6 Hz, 3H); 13C NMR (75 MHz, CDCl3) d 169.9, 139.4, 133.5,
129.1, 128.7, 127.3, 121.7, 74.8, 65.9, 54.4, 41.4, 39.7, 32.3, 31.9,
29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 28.7, 22.6, 14.1; HRMS (ESI) calcd
for [C27H41NO4+H]+: 444.3114, found 444.3121.
Yield: 90%; 1H NMR (300 MHz, CDCl3) d 7.62 (d, J = 0.6 Hz, 1H),
7.27 (d, J = 3.3 Hz, 1H), 6.55 (dd, J = 3.3, 1.5 Hz, 1H), 6.06–5.95 (m,
1H), 5.86 (br s, 1H), 5.52–5.40 (m, 2H), 4.51 (t, J = 3.3 Hz, 2H),
4.33 (dd, J = 9.0, 4.2 Hz, 1H), 4.2–4.1 (m, 2H), 2.12 (d, J = 6.9 Hz,
1H), 2.08 (d, J = 6.9 Hz, 1H), 1.45–12.5 (m, 20H), 0.88 (t,
J = 6.6 Hz, 3H); 13C NMR (75 MHz, CDCl3) d 169.9, 159.1, 157.5,
146.8, 143.9, 140.0, 121.8, 118.8, 112.0, 75.3, 66.1, 54.6, 32.4,
31.9, 29.6, 29.5, 29.4, 29.3, 29.1, 28.7, 22.7, 14.1; HRMS (ESI) calcd
for [C24H37NO5+H]+: 420.2750, found 420.2752.
4.5.8. (S)-4-[(R,E)-1-Hydroxyhexadec-2-enyl]-3-
(cinnamyl)oxazolidin-2-one (5h)
Yield: 94%; 1H NMR (300 MHz, CDCl3) d 7.71 (d, J = 16.2 Hz, 1H),
7.55–7.5 (m, 2H), 7.45–7.38 (m, 3H), 6.43 (d, J = 16.2 Hz, 1H), 6.0–
5.9 (m, 1H), 5.5–5.3 (m, 2H), 5.27 (br s, 1H), 4.48 (t, J = 9.0 Hz, 1H),
4.30 (dd, J = 9.0, 5.1 Hz, 1H), 4.12–4.0 (m, 1H), 2.10 (d, J = 5.1 Hz,
4.5.14. (S)-4-[(R,E)-1-(t-Butyldimethylsilyloxy)but-2-enyl]-3-
cyclopentanecarbonyl)oxazolidin-2-one (13)
To a stirred solution of 12 (2.0 g, 7.37 mmol) in anhydrous THF
(22.1 mL) at À78 °C, under an inert atmosphere of N2, were added

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A. Singh et al. / Bioorg. Med. Chem. 19 (2011) 6174–6181
sodium bis(trimethylsilyl)amide (1 M in THF solution, 44.2 mL).
The solution was stirred for 30 min, and cyclopentanecarbonyl
chloride (3.58 mL, 27.0 mmol) was added at À78 °C. After stirring
at À78 °C for 2 h. The reaction mixture was warmed to room tem-
perature and quenched with aqueous H2O (20 mL). The reaction
product was extracted with ethyl acetate (3 Â 50 mL) and the com-
bined organic layers were washed with brine, dried (MgSO4),
filtered and concentrated in vacuo. The residue was purified by
flash column chromatography on silica gel (n-hexane/
EtOAc = 90:10) to give 13 (2.38 g, 88%) as white oil: 1H NMR
(200 MHz; CDCl3) d 5.81 (dq, J = 14.3, 6.5 Hz, 1H), 5.33 (ddq,
J = 15.3, 7.2, 1.6 Hz, 1H), 4.65 (dd, J = 5.5, 1.3 Hz, 1H), 4.45 (dd,
J = 8.0, 3.0 Hz, 1H), 4.36 (ddq, J = 8.4, 2.8, 1.7 Hz, 1H), 4.16 (t,
J = 8.2 Hz, 1H), 4.0–3.81 (m, 1H), 2.05–1.6 (m, 11H), 0.88 (s, 9H),
À0.026 (s, 3H), À0.044 (s, 3H); 13C NMR (50 MHz; CDCl3) d À5.7,
À4.9, 17.2, 17.3, 25.1, 25.5, 25.5, 29.2, 29.9, 42.4, 58.1, 61.4, 66.8,
128.0, 128.7, 153.1, 176.2; HRMS (ESI) calcd for [C19H33NO4Si+-
Na]+: 390.2077, found 390.2076.
4.6.2. (S)-3-(Cyclopentanecarbonyl)-4-[(R,E)-1-hydroxynon-2-
enyl]oxazolidin-2-one (15b)
Yield: 86%; 1H NMR (200 MHz; CDCl3) d 5.87 (dq, J = 15.7,
7.6 Hz, 1H), 5.42–5.20 (m, 3H), 4.43 (t, J = 8.9 Hz, 1H), 4.25 (dd,
J = 8.9, 5.0 Hz, 1H), 4.00 (dd, J = 8.7, 4.5 Hz, 1H), 2.89–2.57 (m,
1H), 2.05 (q, J = 6.9 Hz, 2H), 1.91–1.61 (m, 8H), 1.26 (br s, 8H),
0.88 (t, J = 6.8 Hz, 3H); 13C NMR (50 MHz; CDCl3) d 13.9, 22.4,
25.5, 25.6, 28.5, 28.6, 29.7, 29.9, 31.4, 32.2, 43.7, 54.6, 66.8, 73.8,
122.3, 138.1, 159.5, 175.5; HRMS (ESI) calcd for [C18H29NO4+Na]+:
346.1994, found 346.1966.
4.6.3. (S)-3-(Cyclopentanecarbonyl)-4-[(R,E)-1-hydroxyundec-2-
enyl]oxazolidin-2-one (15c)
Yield: 88%; 1H NMR (200 MHz; CDCl3) d 5.88 (dq, J = 15.7,
7.6 Hz, 1H), 5.65–5.15 (m, 3H), 4.43 (t, J = 8.9 Hz, 1H), 4.25 (dd,
J = 8.9, 5.0 Hz, 1H), 4.00 (dd, J = 8.6, 4.3 Hz, 1H), 2.94–2.50 (m,
1H), 2.04 (q, J = 6.9 Hz, 2H), 1.95–1.45 (m, 8H), 1.26 (br s, 12H),
0.88 (t, J = 6.4 Hz, 3H); 13C NMR (50 MHz; CDCl3) d 13.9, 22.5,
25.5, 25.6, 28.6, 18.9, 29.1, 29.2, 29.7, 29.8, 31.7, 32.2, 43.7, 54.6,
65.7, 73.8, 122.3, 138.1, 159.6, 175.5; HRMS (ESI) calcd for
[C20H33NO4+Na]+: 374.2307, found 374.2305.
4.5.15. (S)-3-(Cyclopentanecarbonyl)-4-[(R,E)-1-hydroxybut-2-
enyl]oxazolidin-2-one (14)
To a solution of 13 (1.0 g, 2.72 mmol) in anhydrous THF
(8.16 mL) at 0 °C, under an inert atmosphere of N2, was added TBAF
(2.72 mL, 2.72 mmol, 1 M in THF). The reaction mixture was then
stirred for 1 h at 0 °C and warmed to room temperature and stirred
for an additional 2 h before being quenched with saturated aque-
ous NH4Cl solution (5.0 mL). The reaction mixture was extracted
with ethyl acetate (3 Â 20 mL) and combined organic layers were
washed with brine, dried (MgSO4), filtered and concentrated in
vacuo. The residue was purified by flash column chromatography
on silica gel (n-hexane/EtOAc = 75:25) to give 14 (0.68 g, 99%) as
white solid: mp = 42 °C; 1H NMR (200 MHz; CDCl3) d 5.88 (dq,
J = 15.7, 7.6 Hz, 1H), 5.84 (br s, 1H), 5.36 (ddq, J = 14.8, 7.3,
1.6 Hz, 1H), 5.25 (dd, J = 7.3, 4.2 Hz, 1H), 4.41 (t, J = 8.9 Hz, 1H),
4.23 (dd, J = 8.9, 5.0 Hz, 1H), 3.98 (ddd, J = 13.9, 8.9, 4.5 Hz, 1H),
2.84–2.65 (m, 1H), 1.97–1.52 (m, 11H); 13C NMR (50 MHz; CDCl3)
d 17.9, 25.7, 25.7, 29.7, 29.9, 43.8, 54.7, 65.9, 74.0, 124.0, 132.6,
159.9, 175.8; HRMS (ESI) calcd for [C13H19NO4+Na]+: 276.1212,
found 276.1211.
4.6.4. (S)-3-(Cyclopentanecarbonyl)-4-[(R,E)-1-hydroxytridec-2-
enyl]oxazolidin-2-one (15d)
Yield: 89%; 1H NMR (200 MHz; CDCl3) d 5.87 (dq, J = 15.7,
7.6 Hz, 1H), 5.55–5.20 (m, 3H), 4.43 (t, J = 8.9 Hz, 1H), 4.25 (dd,
J = 8.9, 5.0 Hz, 1H), 4.00 (dd, J = 8.6, 4.3 Hz, 1H), 2.87–2.64 (m,
1H), 2.05 (q, J = 6.9 Hz, 2H), 1.92–1.56 (m, 8H), 1.26 (br s, 16H),
0.88 (t, J = 6.4 Hz, 3H); 13C NMR (50 MHz; CDCl3) d 13.9, 22.5,
25.5, 25.6, 28.6, 28.75, 28.9, 29.2, 29.2, 29.4, 29.6, 29.9, 31.7,
32.2, 43.7, 54.6, 65.7, 73.8, 122.3, 138.0, 159.6, 175.5; HRMS (ESI)
calcd for [C22H37NO4+Na]+: 402.2620, found 402.2623.
4.6.5. (S)-3-(Cyclopentanecarbonyl)-4-[(R,E)-3-cyclopentyl-1-
hydroxyallyl]oxazolidin-2-one (15e)
Yield: 78%; mp = 49 °C; 1H NMR (200 MHz; CDCl3) d 5.85 (dd,
J = 14.3, 7.6 Hz, 1H), 5.67 (br s, 1H), 5.49–5.05 (m, 2H), 4.42 (t,
J = 8.9 Hz, 1H), 4.24 (dd, J = 8.9, 5.0 Hz, 1H), 4.00 (dd, J = 9.0,
4.1 Hz, 1H), 2.90–2.59 (m, 1H), 2.58–2.17 (m, 1H), 1.89–1.51 (m,
12H), 1.25 (br s, 4H); 13C NMR (50 MHz; CDCl3) d 24.9, 25.6, 25.7,
29.73, 29.9, 32.6, 32.7, 42.9, 43.8, 54.7, 65.9, 73.9, 120.3, 142.8,
159.4, 175.6; HRMS (ESI) calcd for [C17H25NO4+Na]+: 330.1681,
found 329.9668.
4.6. General procedure for the preparation of the compound 15
To a solution of 14 (1 equiv) in anhydrous dichloromethane
(0.48 M) at room temperature under an inert atmosphere of N2
was added the corresponding olefin (1.2 equiv). Specifically for
the preparation of 15a, 15b, 15c, 15d, 15e, 15f and 15g 1-hexene,
1-octene, 1-nonene, 1-dodecene, methylenecyclopentane, methyl-
enecyclohexane and stylene were used. The reaction mixture was
then stirred for 5 min. Grubbs type II catalyst (benzylidene[1,3-
bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(tricy-
clohexylphosphine)-ruthenium, 5 mol %) was added in one por-
tion, and resulting homogeneous solution was stirred for 24 h at
room temperature. After evaporation, the residue was purified by
flash column chromatography on silica gel (n-hexane/
EtOAc = 75:25, v/v) to give 15 in 75–89% yield.
4.6.6. (S)-4-[(R,E)-3-Cyclohexyl-1-hydroxyallyl]-3-
(cyclopentanecarbonyl)oxazolidin-2-one (15f)
Yield: 75%; mp = 41 °C; 1H NMR (200 MHz; CDCl3) d 5.82 (dq,
J = 15.7, 7.6 Hz, 1H), 5.70 (br s, 1H), 5.42–5.09 (m, 2H), 4.41 (t,
J = 8.9 Hz, 1H), 4.24 (dd, J = 8.9, 4.9 Hz, 1H), 4.00 (ddd, J = 12.9,
8.6, 4.3 Hz, 1H), 2.87–2.62 (m, 1H), 1.97–1.54 (m, 13H), 1.32–
0.98 (m, 6H); 13C NMR (50 MHz; CDCl3) d 25.5, 25.6, 25.8, 29.6,
29.8, 32.2, 40.2, 43.7, 54.6, 65.7, 73.9, 119.8, 143.3, 159.5, 175.5;
HRMS (ESI) calcd for [C18H27NO4+Na]+: 344.1838, found 344.1836.
4.6.7. (S)-3-(Cyclopentanecarbonyl)-4-[(R,E)-1-hydroxy-4-
phenylbut-2-enyl]oxazolidin-2-one (15g)
4.6.1. (S)-3-(Cyclopentanecarbonyl)-4-[(R,E)-1-hydroxyhept-2-
enyl]oxazolidin-2-one (15a)
Yield: 82%; mp = 48 °C; 1H NMR (400 MHz; CDCl3) d 7.65–6.76
(m, 5H), 5.96 (dt, J = 15.5, 7.6 Hz, 1H), 5.71 (dd, J = 9.5, 5.0 Hz,
1H), 5.58 (br s, 1H), 5.41 (dd, J = 10.9, 9.4 Hz, 1H), 4.45 (t,
J = 8.9 Hz, 1H), 4.24 (dd, J = 9.0, 5.2 Hz, 1H), 4.02 (ddd, J = 13.8,
9.5, 5.0 Hz, 1H), 3.63 (dd, J = 15.7, 8.2 Hz, 1H), 3.48 (dd, J = 15.6,
7.1 Hz, 1H), 2.84–2.68 (m, 1H), 1.98–1.49 (m, 8H); 13C NMR
(100 MHz; CDCl3) d 25.7, 25.8, 29.8, 30.1, 34.4, 43.7, 54.8, 66.2,
69.5, 122.7, 126.4, 128.3, 128.7, 137.0, 139.2, 159.0, 175.7; HRMS
(ESI) calcd for [C19H23NO4+Na]+: 352.1525, found 352.1523.
Yield: 88%; 1H NMR (200 MHz; CDCl3) d 5.89 (ddd, J = 20.7, 13.6,
6.5 Hz, 1H), 5.63 (br s, 1H), 5.41–5.23 (m, 2H), 4.43 (t, J = 8.8 Hz,
1H), 4.25 (dd, J = 8.8, 5.0 Hz, 1H), 4.00 (dd, J = 8.7, 4.3 Hz, 1H),
2.87–2.65 (m, 1H), 2.08 (q, J = 6.7 Hz, 2H), 1.94–1.56 (m, 8H),
1.40–1.24 (m, 4H), 0.89 (t, J = 6.8 Hz, 3H); 13C NMR (50 MHz;
CDCl3) d 13.5, 22.8, 25.4, 25.5, 29.5, 29.7, 30.6, 31.7, 43.6, 54.5,
65.6, 73.7, 122.3, 137.7, 159.6, 175.4; HRMS (ESI) calcd for
[C16H25NO4+Na]+: 318.1681, found 318.1683.

A. Singh et al. / Bioorg. Med. Chem. 19 (2011) 6174–6181
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12. (a) Wang, C. Y.; Cusack, J. C., Jr.; Liu, R.; Baldwin, A. S., Jr. Nat. Med. 1999, 5, 412;
(b) Burow, M. E.; Weldon, C. B.; Tang, Y.; Navar, G. L.; Krajewski, S.; Reed, J. C.;
Hammond, T. G.; Clejan, S.; Beckman, B. S. Cancer Res. 1998, 58, 4940.
13. (a) Webb, L. M.; Arnholt, A. T.; Venable, M. E. Mol. Cell. Biochem. 2010, 337, 153;
(b) Modrak, D. E.; Leon, E.; Gold, D. V. Mol. Cancer Res. 2009, 7, 890.
14. Bourbon, N. A.; Sandirasegarane, L.; Kester, M. J. Biol. Chem. 2002, 277, 3286.
15. (a) Mathew, T.; Cavallari, M.; Billich, A.; Bornancin, F.; Nussbaumer, P.; De
Libero, G.; Vasella, A. Chem. Biodivers. 2009, 6, 1688; (b) Antoon, J. W.; Liu, J.;
Gestaut, M. M.; Burow, M. E.; Beckman, B. S.; Foroozesh, M. J. Med. Chem. 2009,
52, 5748; (c) Bai, A.; Szulc, Z. M.; Bielawski, J.; Mayroo, N.; Liu, X.; Norris, J.;
Hannun, Y. A.; Bielawska, A. Bioorg. Med. Chem. 2009, 17, 1840; (d) Struckhoff,
A. P.; Bittman, R.; Burow, M. E.; Clejan, S.; Elliott, S.; Hammond, T.; Tang, Y.;
Beckman, B. S. J. Pharmacol. Exp. Ther. 2004, 309, 523; (e) Antoon, J. W.; Liu, J.;
Ponnapakkam, A. P.; Gestaut, M. M.; Foroozesh, M.; Beckman, B. S. Cancer
Chemother. Pharmacol. 2010, 65, 1191; (f) Lim, H. S.; Oh, Y. S.; Suh, P. G.; Chung,
S. K. Bioorg. Med. Chem. Lett. 2003, 13, 237.
16. (a) Selzner, M.; Bielawska, A.; Morse, M. A.; Rudiger, H. A.; Sindram, D.;
Hannun, Y. A.; Clavien, P. A. Cancer Res. 2001, 61, 1233; (b) Fillet, M.; Bentires-
Alj, M.; Deregowski, V.; Greimers, R.; Gielen, J.; Piette, J.; Bours, V.; Merville,
M.-P. Biochem. Pharmacol. 2003, 65, 1633.
17. (a) Karasavvas, N.; Erukulla, R. K.; Bittman, R.; Lockshin, R. Eur. J. Biochem. 1996,
236, 729; (b) Jarvis, W. D.; Fornari, F. A., Jr.; Traylor, R. S.; Martin, H. A.; Kramer,
L. B.; Erukulla, R. K.; Bittman, R.; Grant, S. J. Biol. Chem. 1996, 271, 8275; (c)
Chang, Y. T.; Choi, J.; Ding, S.; Prieschl, E. E.; Baumruker, T.; Lee, J. A.; Chung, S.
K.; Schultz, P. G. J. Am. Chem. Soc. 2002, 124, 1856.
18. (a) Chun, J.; He, L.; Byun, H.-S.; Bittman, R. J. Org. Chem. 2000, 65, 7634; (b)
Bielawska, A.; Linardic, C. M.; Hannun, Y. A. J. Biol. Chem. 1992, 267, 18493; (c)
Bielawska, A.; Greenberg, M. S.; Perry, D.; Jayadev, S.; Shayman, J. A.; McKay, C.;
Hannun, Y. A. J. Biol. Chem. 1996, 271, 12646.
19. (a) Macchia, M.; Barontini, S.; Bertini, S.; Di Bussolo, V.; Fogli, S.; Giovannetti,
E.; Grossi, E.; Minutolo, F.; Danesi, R. J. Med. Chem. 2001, 44, 3994; (b) Macchia,
M.; Bertini, S.; Fogli, S.; Giovannetti, E.; Minutolo, F.; Rapposelli, S.; Danesi, R. Il
Farmaco 2003, 58, 205.
20. Ha, H.-J.; Hong, M. C.; Ko, S. W.; Kim, Y. W.; Lee, W. K.; Park, J. Bioorg. Med.
Chem. Lett. 2006, 16, 1880.
21. Rerek, M. E.; Chen, H.-C.; Markovic, B.; Wyck, D. V.; Garidel, P.; Mendelsohn, R.;
Moore, D. J. J. Phys. Chem. B 2001, 105, 9355.
Acknowledgment
This work was supported by the National Research Foundation
(the Center for Bioactive Molecular Hybrides, 2011-0012379 and
2010-0008424 to H.J.H., and NRF-2010-0005538 and NRF-2009-
0081956 to W.K.L.).
References and notes
1. (a) Kolter, T.; Sandhoff, K. Chem. Soc. Rev. 1996, 25, 371; (b) Dickson, R. C. Ann.
Rev. Biochem. 1998, 67, 27; (c) Kolter, T.; Sandhoff, K. Angew. Chem., Int. Ed.
1999, 38, 1532; (d) Brodeser, S.; Sawatzki, P.; Kolter, T. Eur. J. Org. Chem. 2003,
2021; (e) Liao, J.; Tao, J.; Lin, G.; Liu, D. Tetrahedron 2005, 61, 4715; (f)
Ogretmen, B.; Hannun, Y. A. Nat. Rev. Cancer 2004, 4, 604; (g) Pyne, N. J.; Pyne, S.
Nat. Rev. Cancer 2010, 10, 489.
2. (a) Pettus, B. J.; Chalfant, C. E.; Hannun, Y. A. Biochim. Biophys. Acta 2002, 1585,
114; (b) Radin, N. S. Bioorg. Med. Chem. 2003, 11, 2123; (c) Reynolds, C. P.;
Maurer, B. J.; Kolesnick, R. N. Cancer Lett. 2004, 206, 169; (d) Hannun, Y. A.;
Obeid, L. M. Nat. Rev. Cell Biol. 2008, 9, 139; (e) Saddoughi, S. A.; Song, P.;
Ogretmen, B. Subcell. Biochem. 2008, 49, 413; (f) Schiffmann, S.; Sandner, J.;
Birod, K.; Wobst, I.; Angioni, C.; Ruckhaberle, E.; Kaufmann, M.; Ackermann, H.;
Lotsch, J.; Schmidt, H.; Geisslinger, G.; Grosch, S. Carcinogenesis 2009, 30, 745.
3. (a) Hannun, Y. A. J. Biol. Chem. 1994, 269, 3125; (b) Hannun, Y. A. Science 1996,
274, 1855; (c) Sawai, H.; Hannun, Y. A. Chem. Phys. Lipids 1999, 102, 141.
4. Hannun, Y. A.; Luberto, C.; Argraves, K. M. Biochemistry 2001, 40, 4893.
5. Cifone, M. G.; Maria, R. D.; Roncaioli, P.; Rippo, M. R.; Azuma, M.; Lanier, L. L.;
Santoni, A.; Testi, R. J. Exp. Med. 1994, 180, 1547.
6. Kim, M.; Linardic, C.; Obeid, L.; Hannun, Y. J. Biol. Chem. 1991, 266, 484.
7. Haimovitz-Friedman, A.; Kan, C. C.; Ehleiter, D.; Persaud, R. S.; McLoughlin, M.;
Fuks, Z.; Kolesnick, R. N. J. Exp. Med. 1994, 180, 525.
8. Kinloch, R. A.; Treherne, J. M.; Furness, L. M.; Hajimohamadreza, I. Trends
Pharmacol. Sci. 1999, 20, 35.
9. (a) Rajewski, R. A.; Kosednar, D. G.; Matches, T. A.; Wong, O. S.; Burchett, K.;
Thakker, K. J. Pharm. Biomed. Anal. 1995, 13, 247; (b) Sachs, C. W.; Safa, A. R.;
Harrison, S. D.; Fine, R. L. J. Biol. Chem. 1995, 270, 26639.
10. Cuadros, R.; Montejo de Garcini, E.; Wandosell, F.; Faircloth, G.; Fernández-
Sousa, J. M.; Avila, J. Cancer Lett. 2000, 152, 23.
22. Van de Loosdrecht, A. A.; Beelen, R. H. J.; Ossenkoppele, G. J.; Broekhoven, M.
G.; Langenhuijsen, M. M. A. C. J. Immunol. Methods 1994, 174, 311.
23. Dagan, A.; Wang, C.; Fibach, E.; Gatt, S. Biochim. Biophys. Acta 2003, 1633, 161.
24. Singh, A.; Kim, B.; Lee, W. K.; Ha, H.-J. Org. Biomol. Chem. 2011, 9, 1372.
25. Chatterjee, A. K.; Grubbs, R. H. Angew. Chem., Int. Ed. 2002, 41, 3172.
11. (a) Yun, J. M.; Sin, T. B.; Hahm, H. S.; Lee, W. K.; Ha, H.-J. J. Org. Chem. 2003, 68,
7675; (b) Kim, J.; Kim, Y.-W.; Inagakic, Y.; Hwanga, Y.-A.; Mitsutakec, S.; Ryue,
Y.-W.; Lee, W.-K.; Ha, H.-J.; Park, C.-S.; Igarashi, Y. Bioorg. Med. Chem. 2005, 13,
3475.