The convergent total synthesis of cytotoxic homospisulosine and its 3-epi-analogue

Article abstract of DOI:10.1016/j.tetasy.2015.11.001

A common strategy for the total synthesis of cytotoxic homospisulosine 7 and its 3-epi-analogue 10 was achieved in a stereoconvergent manner, from 3,5-di-O-benzyl-d-xylofuranose with the efficient use of rearranged products 14-17. The key transformations of this approach are [3,3]-heterosigmatropic rearrangements of allylic substrates 18 and 19 to establish a stereogenic amino group and regioselective intramolecular cyclization to form common oxazolidinones 28 and 29. Elongation of the side chain was accomplished via a Wittig reaction. Several compounds were evaluated for their in vitro antiproliferative/cytotoxic activity by the MTT method employing five different human cancer cell lines (Jurkat, MDA-MB-231, MCF-7, HCT-116 and Caco-2). The obtained data revealed that homospisulosine 7 exhibited the most significant inhibitory effects among all of the screened compounds on the panel of the tested cell lines with IC₅₀ values comparable to those of conventional anticancer agents such as cisplatin and etoposide.

Full text of DOI:10.1016/j.tetasy.2015.11.001

Tetrahedron: Asymmetry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
The convergent total synthesis of cytotoxic homospisulosine
and its 3-epi-analogue
a
b
b
Kvetoslava Stanková a, Miroslava Martinková a, , Jozef Gonda , Martina Bago , Martina Pilátová ,
⇑
Gabriela Gönciová b
a Institute of Chemical Sciences, Department of Organic Chemistry, P.J. Šafárik University, Moyzesova 11, 040 01 Košice, Slovak Republic
b Institute of Pharmacology, Faculty of Medicine, P.J. Šafárik University, SNP 1, 040 66 Košice, Slovak Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
A common strategy for the total synthesis of cytotoxic homospisulosine 7 and its 3-epi-analogue 10 was
achieved in a stereoconvergent manner, from 3,5-di-O-benzyl-D-xylofuranose with the efficient use of
Received 10 September 2015
Accepted 5 November 2015
Available online xxxx
rearranged products 14–17. The key transformations of this approach are [3,3]-heterosigmatropic rear-
rangements of allylic substrates 18 and 19 to establish a stereogenic amino group and regioselective
intramolecular cyclization to form common oxazolidinones 28 and 29. Elongation of the side chain
was accomplished via a Wittig reaction. Several compounds were evaluated for their in vitro antiprolif-
erative/cytotoxic activity by the MTT method employing five different human cancer cell lines (Jurkat,
MDA-MB-231, MCF-7, HCT-116 and Caco-2). The obtained data revealed that homospisulosine 7 exhib-
ited the most significant inhibitory effects among all of the screened compounds on the panel of the
tested cell lines with IC50 values comparable to those of conventional anticancer agents such as cisplatin
and etoposide.
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
sphingolipids, and both 1 and 2 have been found to inhibit protein
kinase C.1h,7 Moreover, sphingoid base 2 represents a metabolic
Sphingolipids, a conspicuous class of natural products including
sphingomyelins, cerebrosides and more complex glycosphin-
golipids are essential components of eukaryotic cells. Besides
pivotal structural roles in the membrane construction, they also
serve as a source for bioactive metabolites such as sfingosine-1-
phosphate, sphingosine and ceramide, which are involved in vari-
ous significant cell signalling pathways and processes.1 Membrane
sphingolipids were discovered as far back as 1884 by Thudichum2
and over the past 20 years much has been learned about their chem-
istry and biology.1 The principal backbone of sphingolipids is based
on an architecturally related class of lipophilic compounds that are
very often termed the sphingoid bases.1g This aforementioned
family involves the aliphatic amino alcohols with long
hydrocarbon side chains where three C18 derivatives, sphinganine
switch between sphingosine-1-phosphate, which mediates cell
proliferation and angiogenesis and ceramide, which is implicated
in signalling processes leading to apoptosis and also inhibits
proliferation.1d It should be noted that 2 is not easily obtainable
from natural sources and is prepared via chemical synthesis, in
contrast to
D-ribo-phytosphingosine 3 (Fig. 1), which is readily
accessible on an industrial scale through fermentation processes8,9
and represents the most abundant member of the phytosphingosine
family, originally isolated from the mushroom Amanita muscaria.10
Natural sphinganine 1, sphingosine 2 and other related members
of the mammalian sphingolipid family have a (2S,3R)-configuration,
but sphingoid bases produced by invertebrates and microbes pos-
sess various stereochemical arrangements.3b,11 In addition to the
aforementioned configurational heterogeneity, the natural sources
extend further the structural and compositional variations of
sphingoid bases, including simpler 1-deoxysphinganines such as
spisulosine 4 (Fig. 1) and its several related analogues lacking the
1,3
D
-erythro-sphingosine 23b,4 and
D-ribo-phytosphingosine
3,3b,4b,5 are predominant in Nature (Fig. 1). Among them, 1 has
been found in many organisms as an early intermediate in the de
novo pathway1,6 leading to sphingolipids, including 2 and 3.
C-1 hydroxy functionality.3b (2S,3R)-2-Aminooctadecan-3-ol
4
D
-erythro-Sphingosine
2
is the major core unit in mammal
(known as spisulosine or also referred to as ES-285) is a marine
bioactive molecule produced by the clam Spisula polynyma (syn.
Mactromeris polynyma) together with its further side-chain
analogues 5 and 6.12a,b It has been found that 412 and a number of
⇑
Corresponding author. Tel.: +421 55 2342329; fax: +421 55 6222124.
E-mail address: miroslava.martinkova@upjs.sk (M. Martinková).
http://dx.doi.org/10.1016/j.tetasy.2015.11.001
0957-4166/Ó 2015 Elsevier Ltd. All rights reserved.
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2
K. Stanková et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
NH2
NH2 OH
OH
NH2
HO
HO
HO
12
12
12
OH
OH
D-ribo -phytosphingosine 3
D-erythro -sphingosine 2
1
D-erythro-sphinganine
NH2
NH2
X
3
n
OH
Y
Y= (3R)-OH: xestoaminol C 8
Y= (3S)-OH: 3-epi-xestoaminol C
X = H, n = 12: spisulosine 4
X = H, n = 13:
9
5
X = H, n = 14: 6
X = CH3, n = 12: homospisulosine
7
Figure 1. Structures of the natural sphingoid bases and related deoxysfinganines.
its unnatural analogues, including homospisulosine 7,12c exhibit
potent in vitro cytotoxicity against several cancer cell lines such as
L1210,12a P-388,12a,c A-549,12a,c HT-29,12a,c MeL-2812a,c and
DU-14512c with IC50 values down to the nanomolar levels, as well
as against certain human malignant solid tumours and leukaemias/
lymphomas.12a,c Moreover, spisulosine 4 was screened in vivo for
anticancer activity on xenograft models of human PC-3 and MRI-
H-121 solid tumour types and in both cases significantly reduced
their size.12a Next, Díaz-Laviada et al. revealed its ability to inhibit
the proliferation in the prostate tumour PC-3 and LNCaP cell lines
with IC50 in the micromolar ranges via intracellular ceramide accu-
mulation and activation of the atypical protein kinase C (PKCf).13 In
another study reported by Avila et al.,14 it was shown that 4 inhibits
cell proliferation in Vero cells (a line of African green monkey kidney
cells) by the disassembly of actin stress fibres through Rho signalling
pathway.
novel antiproliferative/cytotoxic agents. The simpler, but unique
structure possessing the (2S,3R)-configured amino alcohol unit
with the neighbouring long alkyl chain represents
a motif
common to the most abundant mammalian sphingoids (Fig. 1)
which, when combined with the significant biological findings
and the limited availability from natural sources, has made
spisulosine 4 an attractive target for total synthesis.15,16,20,21
Reported approaches leading to the construction of 4 in the form
of a free amine utilize either chiral pool starting materials such
as
-glyceraldehyde,20b (S)-Garner’s aldehyde,20d
Boc-
N-tert-butylsulfinyl
imines,20c
2,3-O-isopropylidene-
D
L
-alanine,20g,k N-
L
-alaninal,15 ethyl (2R,3R)-2-azido-3-hydroxyoctadecanoate16
and enantiomerically pure 2-acylaziridines,20a which were
chosen for the construction of the N-Boc-spisulosine, or use
simple achiral molecules such as hexadecan-1-ol,20e (E)-pent-
3-en-1-yl sulfamate,20f palmitaldehyde,20h heptadec-1-ene20i
Recently, Delgado et al.15 reported a stereoselective synthesis of
spisulosine and its analogues as probes for ceramide synthase
activity profiling in intact cells. Among the prepared compounds
(eight derivatives), spisulosine 4, its 3-epi-analogue and
3-epi-4,5-dehydrospisulosine were found to be the most suitable
ones and could potentially be useful for the study of the role of dif-
ferent ceramide synthases and their resulting products in cell fate.
Bittman et al.16 demonstrated that spisulosine 4 selectively blocks
sphingosine kinase (SphK1), inducing its proteasomal degradation
and reducing DNA synthesis in human pulmonary arterial smooth
muscle cells. Padrón et al. revealed that 4 and several of its deriva-
and propionaldehyde20j in which
a
Sharpless asymmetric
dihydroxylation,20e,i Overman rearrangement,20i copper-catalyzed
asymmetric intramolecular alkene aziridination of sulfamate,20f
asymmetric Henry reaction20h and proline-catalyzed
a-amination
followed by In-mediated allyaltion20j are involved as the key
transformations (Fig. 2). To the best of our knowledge, there have
been published six independent syntheses of 820g,22,23 (Fig. 2) in
the form of
a
free base or its corresponding N,O-diacetate
-alanine,20g,22a tert-butyl (E)-but-2-enoate,22b
-glucose22d and chiral 2-acylaziridine23
from the chiral
derivative from
L
dodec-2-ynal,22c
D
a
and only one construction of molecule
9
tives behaved as selective CK1e inhibitors and displayed significant
2-acylaziridine.23
antiproliferative activity against different human tumour cell lines
(HBL-100, HeLa, SW1573, T-47D, WiDr).17 In addition to the long
chain deoxysphingoid bases discussed above, marine organisms
produce also a wide variety of their truncated analogues such as
xestoaminol C 8 together with its 3-epimer 9 (Fig. 1). Xestoaminol
C has been isolated independently from two natural sources; in
1990 from the Fijian sponge Xestospongia sp.18a and in 2001 was
found in the tunicate Pseudodistoma obscurum.18b Compound 8
has been shown to be an inhibitor of reverse transcriptase.18a
Recently, Keyzers et al. reported the isolation of 3-epi-
xestoaminol C 9 from the New Zealand brown alga Xiphophora
chondrophylla, together with its full structure determination,
including the (2S,3S)-absolute configuration, by utilizing
Mosher’s method.19 Their biological screening revealed that 9
exhibited moderate cytotoxicity with a broad range of antimicro-
bial activity.19
Our previous success with the total synthesis of jaspine
B, its five stereoisomers24a–c and eight members of the
phytosphingosine family24 from simple carbohydrates, by employ-
ing [3,3]-heterosigmatropic rearrangements to provide direct
access to the required vicinal amino alcohol scaffolds, turned our
attention to the group of deoxysphinganines. In light of the above
investigations, we envisioned applying this strategy to the synthe-
sis of two unnatural cytotoxic sphingoid-type bases, homospisu-
losine 712c and its 3-epi-analogue 10. So far, just one synthesis of
7 has been described in the literature, which utilized (S)-2-
aminobutanoic acid as a chiral pool.12c On the other hand, for its
3-epimer 10, no synthesis has been reported.
2. Results and discussion
2.1. Chemistry
Due to the close structural relationship of 412 and its known
natural related analogues3b,18,19 with the bioactive sphingolipid
metabolites,1d such compounds as well as their synthetic
derivatives are expected to be promising lead structures for
The clear structural features of the aforementioned deoxysphin-
golipids 7 and 10, led us to disconnect their carbon framework into
two parts, the highly functionalized aminopolyols 11 and 12 and
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K. Stanková et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
3
2,3-O-isopropylidene-
D-glyceraldehyde
chiral
2-acylaziridine
palmitaldehyde
and propionaldehyde
(S)-Garner aldehyde
NH2
NH2
L-alanine
heptadec-1-ene
D-glucose
L-alanine
and N-Boc-L-alaninal
9
13
OH
8
OH
4
(E)-pent-3-ene-1-yl
sulfamate
N-tert-butylsulfinyl
imine
dodec-2-ynal
tert-butyl (E)-but-2-enoate
hexadecan-1-ol
ethyl (2R,3R)-2-azido-3-hydroxy-
octadecanoate
Figure 2. Reported syntheses of spisulosine 4 and xestoaminol C 8.
the aliphatic phosphonium salt 13,25 which we anticipated would
be coupled by the well-established Wittig reaction (Scheme 1).
We expected to construct 11 and 12 from the corresponding rear-
ranged products 14, 16 and 15, 17, respectively, in which the novel
stereogenic centre with an amino functionality can be installed via
[3,3]-sigmatropic rearrangements of the chiral allylic substrates 18
corresponding allylic alcohol 2528 (98%, Scheme 2). Its NMR data
were consistent with the literature values, but the specific rotation
was opposite in sign and its magnitude was different {([a]
25 = +4.2
D
(c 1.28, CHCl3); lit.28
[
a]
24 = ꢀ38.8 (c 1.24, CHCl3)}. The next objec-
D
tive was the transformation of 25 into the required aza-Claisen
substrates 18 and 19 for the key rearrangement processes. Thus,
and 19. For their preparation,
D-xylose was selected as a starting
a two-step sequence involving mesylation/KSCN treatment
point.
resulted in the formation of thiocyanate 18 (86%). On the other
hand, the application of standard conditions such as trichloroace-
tonitrile and NaH to alcohol 25 gave imidate 19 (Scheme 3) in
nearly quantitative yield, which was used in all rearrangement
reactions in the crude form.
As shown in Scheme 2, our synthesis began with the gram-scale
preparation of the known 3,5-di-O-benzyl-
D
-xylofuranose 20,26
obtained from
D-xylose in three steps, by applying slightly
modified literature procedures.26,27 The obtained material had 1H
NMR data in accordance with those reported.26 The subsequent
Wittig olefination (Ph3P = CHCO2Et, CH2Cl2, benzoic acid) of lactol
Having completed the preparation of both allylic products 18
and 19, the [3,3]-heterosigmatropic rearrangements for the instal-
lation of the new C–N bond were the next focus (Scheme 3). The
prepared thiocyanate 18 was rearranged in n-heptane at 70 °C
and 90 °C using both conventional thermal conditions and micro-
wave heating, to provide the corresponding isothiocyanates 14
and 15 as an easily separable mixture of diastereoisomers in good
yields (79–81%) and with acceptable stereoselectivity (14:15 =
84:16, Table 1, entry 1). It should be noted that during these
experiments we recovered the starting material 18 in approxi-
mately 10–14% yields after chromatographic separation, which
was repeatedly utilized. The prolonged heating turned out to be
ineffective and had practically no influence on the overall yield
of the rearranged products 14 and 15. On the other hand, the
microwave accelerated Overman rearrangement29 of 19, which
was carried out in o-xylene at 110 °C, 130 °C and 140 °C, in the
20 provided a barely separable mixture of
a,b-unsaturated esters
(E)-21:(Z)-22 = 80:20 as evidenced by 1H NMR spectroscopy, in
90% combined yield (Scheme 2). Repeated chromatography of the
small amounts of the obtained mixture of olefins furnished only
(E)-21 in pure form as an analytical sample. The presence of the
trans double bond at C-2/C-3 in (E)-21 was unambiguously
confirmed from the vinyl proton coupling constant value
(J3,2 = 15.6 Hz).
To continue the synthesis, the syn-1,3-diol relationship in the
(E)-21/(Z)-22 mixture was protected as an acetonide (2,2-DMP,
p-TsOH, CH2Cl2) and facilitated the separation of two isopropyli-
dene derivatives (Z)-23 and (E)-24, which were isolated in 18%
and 70% yields, respectively. Reduction of the ester functionality
in the major geometrical isomer (E)-24 was achieved with
diisobutylaluminum hydride in CH2Cl2 and furnished the
30
presence of K2CO3 produced the desired trichloroacetamides 16
O
R1
OH
O
R2
NR3
R1
Wittig
P+Ph3Br-
D-xylose
+
13
12
OH
OH R2
OH
1325
11, R1 = H, R2 = Et
12, R1 = Et, R2 = H,
R3 = Bn
7, R1 = NH2, R2 = H
10
1
, R = H, R2 = NH2
O
O
O
O
[3,3]
R
R2
R1
OBn OBn
OBn OBn
14, R1 = NCS, R2 =H
15, R1 = H, R2 = NCS
18, R = SCN
, R = O(C=NH)CCl3
19
16, R1 = NHCOCCl , R2 = H
3
17, R1 = H, R2 = NHCOCCl
3
Scheme 1. Synthetic plan leading to deoxysphingolipids 7 and 10.
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K. Stanková et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
O
OH
OH
O
O
CO2Et
OH
BnO
BnO
a
BnO
BnO
b
c
CO2Et
BnO
OH
OBn
OBn
2026
23
21
22
(Z)- , 18%
(E)- :(Z)-
= 80:20, 90%
+
O
O
O
O
BnO
OH
CO2Et
OBn
OBn
25,28 98%
24
(E)- , 70%
Scheme 2. Reagents and conditions: (a) Ph3P@CHCO2Et, CH2Cl2, benzoic acid, rt; (b) 2,2-DMP, p-TsOH, CH2Cl2, rt; (c) DIBAl-H, CH2Cl2, ꢀ15 °C.
O
O
O
O
O
CCl3
SCN
a
b
25
25
OBn OBn
NH
OBn OBn
19
d
18, 86%
c
2
O3 1O
O
O
O
O
O
O
+
5
1`
+
4
6
OBn OBn NCS
OBn OBn NCS
OBn OBn NHCOCCl
OBn OBn NHCOCCl3
3
14
15
17
16
Scheme 3. Reagents and conditions: (a) (i) MsCl, Et3N, CH2Cl2, 0 °C ? rt; (ii) KSCN, MeCN, 5 °C ? rt; (b) CCl3CN, NaH, THF, 0 °C; (c) Table 1; (d) Table 2.
methodology. The conformers within a 3 kcal/mol energy range
Table 1
were then optimized using B3LYP/6-31G(d) for a more accurate
description of the conformer distribution. The low energy conform-
ers of the transition states were later fully optimized using
B3LYP/6-31+G(d,p). It is worth noting that the rearranged part
and the –CH2OBn group have an equatorial arrangement, while
the OBn group is axial in all of the low energy conformers. The
potential energy surface scans were used to explore the conforma-
tional space of the reactant and products thiocyanate 18 and isoth-
iocyanates 14 and 15 with the later full optimization using the same
level of theory; however we only focused on the energy differences
between TSs as being crucial for the explanation of the observed
diastereoselectivities. The geometries of the transition states were
optimized using B3LYP/6-31+G(d,p) with the Jaguar 7.7 pro-
gramme.31 The nature of the vacuum B3LYP transition states was
verified with frequency calculations, yielding only one large imag-
inary frequency. Harmonic zero-point energy corrections at
B3LYP/6-31+G(d,p), obtained from the frequency calculations of
the vacuum transition states, were applied to the transition-state
energies. Single-point energies were computed by the B3LYP den-
sity functional method and the cc-pvTZ basis set. The solvent effect
was taken into account via a single-point calculation in a dielectric
continuum representing n-heptane as the solvent. A standard PBF
solvation model was applied as implemented in the Jaguar 7.7 pro-
gramme.31 The [3,3]-sigmatropic rearrangement of 18 occurs via
transition states TS1 and TS2 with relative free energies 0 and
0.94 kcal/mol (Fig. 3). This process is concerted but asynchronous.
From the calculations, for the pathway 18?TS1?14, the activation
energy was found to be 1.135 kcal/mol lower than for the pathway
18?TS2?15. Thus, the predicted diastereomeric ratio of 14:15 at
70 °C was 80:20. These results are in very good agreement with
the experimental data (14:15 = 84:16, Table 1, entry 1) with the
correct prediction of isothiocyanate 14 as being the predominant
diastereoisomer.
[3,3]-Sigmatropic rearrangement of thiocyanate 18
Entry
Thiocyanate
Conditionsa
, 70 °C
MW, 70 °C
, 90 °C
MW, 90 °C
Time (h)
Ratiob 14:15
Yieldc (%)
1
2
3
4
18
18
18
18
D
6
2
2
1
84:16
81:19
82:18
70:30
81
81
79
79
D
a
b
c
In n-heptane.
Ratio in the crude reaction mixtures. Determined by 1H NMR analysis.
Isolated combined yields.
and 17 in excellent yields (88–94%), but no diastereoselectivity
was observed (Table 2).
In order to rationalize the observed stereoselectivity in the [3,3]-
sigmatropic rearrangement of thiocyanate 18, high-level density
functional theory (DFT) calculations, including electron correlation
effects, were carried out. A thorough conformational search was
performed on all transition states, although only the lowest energy
conformers are discussed herein. The potential energy surface scans
were used to explore the conformational space of transition states
TS1 and TS2 with the constrained transition bond lengths C–N
and C–S. The systematic conformational search with rotation
around the single bonds was performed using semiempirical PM3
Table 2
Overman rearrangement of imidate 19
Entry
Imidate
Conditionsa
Time (h)
Ratiob 16:17
Yieldc (%)
1
2
3
19
19
19
MW, 110 °C
MW, 130 °C
MW, 140 °C
6
1
0.5
55:45
57:43
54:46
88
94
90
a
b
c
In o-xylene, in the presence of K2CO3.
Ratio in the crude reaction mixtures. Determined by 1H NMR analysis.
Isolated combined yields.
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K. Stanková et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
5
Figure 3. Transition structures for the rearrangement 18 ? [TS1]/[TS2] ? 14 + 15. Relative energies of transition states (in kcal/mol) and bond distances (in Å) are shown.
Having constructed scaffolds, which possess the required vicinal
amino alcohol motif, we were able to determine the stereochem-
istry of the newly constructed stereocentre with the nitrogen func-
tionality. The minor isothiocyanate 15 was isolated as the
crystalline derivative and after recrystallization from n-hexane, fur-
nished suitable crystals for X-ray structure determination. The crys-
tallographic analysis of 15 (Fig. 4) unambiguously confirmed that
the carbon atom bearing an NCS group had an (R)-configuration,
revealing that the major diastereoisomer of the rearrangement,
compound 14, must be the (10S)-isomer (for the corresponding
numbering, see Scheme 3). Although both Overman’s products 16
and 17 were prepared as solid compounds, their prepared crystals
were inadequate for X-ray diffraction analysis. In order to assign
the correct stereochemistry in both stereoisomers 16 and 17, the
chemical correlations were therefore carried out (Scheme 4).
Thus, deprotection of the acetonide fragment in 14 with p-TsOH
in MeOH followed by the spontaneous intramolecular nucleophilic
addition of the regenerated alcohol functionality to an –NCS moi-
ety afforded the corresponding cyclic thiocarbamate 26 in 93%
yield. This transformation proceeded exclusively in a regioselective
manner and no trace of the 7-membered 1,3-oxazepane-2-thione
was detected in the crude reaction mixture (judged by 1H
NMR spectroscopic analysis). In a parallel fashion, the minor
Figure 4. ORTEP structure of 15 showing the crystallographic numbering.
diastereoisomer 15 was elaborated into the desired derivative 27
(97%, Scheme 4). Compounds 26 and 27 were then treated with
mesitylnitrile oxide to furnish the key oxazolidinones 28 (98%)
on the stereocentre installed by the studied [3,3]-sigmatropic
rearrangements.
and 29 (94%), respectively. In a similar manner, both trichloroac-
etamides 16 and 17 were modified into the common intermediates
28 and 29. This included the opening of the 1,3-O-isopropylidene
skeleton in 16 and 17 using the same reaction conditions as in
the case of isothiocyanates 14 and 15 to give alcohols 30 (98%)
Since we had already assigned the C–N configuration of all of
the rearranged products and built up the common scaffolds 28
and 29, the remaining task of our synthetic route was their cou-
pling to the long hydrocarbon side chain 13, which was achieved
using the approach shown in Schemes 5 and 6.
The catalytic hydrogenation of 28 under heterogeneous Pd
(OH)2/Pd/C/H2 conditions removed two O-benzyl moieties and the
saturated the vinyl residue to furnish the corresponding triol 11
in 79% yield (Scheme 5). Oxidative fragmentation of 11 with NaIO4
furnished the corresponding aldehyde, which was then coupled
with the non-stabilized ylide derived from the known salt 13,25
and 31 (96%). Their treatment with
a base (DBU) caused
intramolecular cyclization to provide the desired derivatives 28
and 29 in 86% and 95% yields, respectively (Scheme 4). Spectro-
scopic data and the specific rotation of oxazolidinone 28 prepared
from the slightly preferred trichloroacetamide 16 were in excellent
agreement with those obtained for 28 produced from the highly
prevalent isothiocyanate 14. Consequently, both diastereoisomers
14 and 16 must possess the same stereochemical arrangement
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K. Stanková et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
O
S
OH
OH
OH
OH
O
O
a
a
a
b
b
c
c
NH
14
15
NH
16
17
OBn OBn NHCOCCl3
OBn OBn
OBn OBn
30, 98%
26
28
, 93%
S
O
OH
OH
OH
O
OH
O
a
NH
NH
OBn OBn NHCOCCl
OBn OBn
27, 97%
OBn OBn
3
31, 96%
29
Scheme 4. Reagents and conditions: (a) p-TsOH, MeOH, rt; (b) mesitylnitrile oxide, MeCN, rt, 98% for 28 from 26, 94% for 29 from 27 (c) DBU, CH2Cl2, 0 °C ? rt, 86% for 28
from 30, 95% for 29 from 31.
by employing freshly prepared LHMDS as a base.32 This process
resulted in the formation of a barely separable mixture of olefins
32 (Z:E ꢁ 82:18, determined by 1H NMR) in 78% isolated yield (over
two steps). Repeated chromatography of the small amount of such
mixture afforded only (Z)-32 in the pure form. The counterpart for
the aforementioned Wittig reaction, the corresponding
phosphonium salt 13, was synthesized from the commercially
available 1-bromotetradecane (Ph3P, reflux, 24 h) in 79% yield
(see, Section 4.1.22). The spectroscopic data of 13 matched the val-
ues present in the literature for the same compound.25a
The subsequent catalytic hydrogenation of 32 (H2, 5% Rh in
Al2O3)15 provided the requisite saturated derivative 33 (75%)
whose final deprotection (2 M NaOH aq, EtOH) completed the syn-
thesis of homospisulosine 7 (91%, Scheme 5). Our spectroscopic
data, which were found for 7, especially 13C NMR values, showed
differences in comparison to those previously reported.12c It should
be noted that no optical rotation data or a melting point have been
published for 7.
was due to the presence of an unprotected oxazolidinone skeleton.
To overcome this problem, we decided to block the nitrogen atom
of the cyclic carbamate moiety in 29 with a benzyl group (BnBr,
NaH) to furnish 34 in 95% yield (Scheme 6). Its chemoselective
deprotection resulted in the formation of the corresponding triol
12 (83%) whose oxidative cleavage (NaIO4) followed by olefination
(13, LHMDS, THF) afforded a mixture of alkenes 35 (Z:E ꢁ 81:19,
determined by 1H NMR analysis) with very good yield (82%). The
subsequent catalytic hydrogenation (H2, 10% Pd/C, 36% HCl, EtOH)
of 35 removed the double bond as well as the benzyl residue to
give 36 (50%) During this reaction we also recovered smaller
amounts (approximately 10%) of the partially reduced compound
without a double bond. Prolonged heating increased the produc-
tion of the unidentified side products. A final hydrolysis under
basic conditions gave the requisite 3-epi-analogue 10 in 92% yield
(Scheme 6).
2.2. Antiproliferative/cytotoxic activity
To obtain 3-epi-analogue 10, our initial attempts to achieve the
coupling between aldehyde generated from 29, using the same
reaction conditions as described in Scheme 5, and salt 13 turned
out to be ineffective: the corresponding olefins were obtained in
a maximum yield of approximately 32%. We reasoned that this
The final sphingoid bases 7, 10 and also several intermediates of
the developed synthetic strategy compounds 14, 15, 26–29, (Z)-32,
33 and 36 (Table 4) were screened for the antiproliferative/cyto-
toxic activities against five different human cancer cell lines;
O
O
O
NH2
OH
O
d
b
c
a
NH
HN
O
HN
O
28
C13H27
13
OH
7, 91%
OH
OH
11, 79%
C15H31
, 75%
33
32, 78%
Scheme 5. Reagents and conditions: (a) H2, Pd(OH)2/C, 10% Pd/C, EtOH, rt; (b) (i) NaIO4, MeOH/H2O (1:1), rt; (ii) 13,25 LHMDS,32 THF, rt; (c) H2, 5% Rh in Al2O3, EtOAc, rt; (d)
2 M NaOH aq, EtOH, reflux.
O
O
O
O
OBn
O
OH
O
BnN
O
HN
O
NBn
NBn
a
b
c
d
C13H27
29
C15H31
OBn OBn
34
OH
OH
12, 83%
36, 50%
35, 82%
, 95%
e
NH2
13
OH
10
, 92%
Scheme 6. Reagents and conditions: (a) BnBr, NaH, TBAI, DMF, 0 °C ? rt; (b) H2, 10% Pd/C, EtOH, rt; (c) (i) NaIO4, MeOH/H2O (1:1), rt; (ii) 13,25 LHMDS,32 THF, rt; (d) H2, 10%
Pd/C, EtOH, 36% HCl, 60 °C; (e) 2 M NaOH aq, EtOH, reflux.
Please cite this article in press as: Stanková, K.; et al. Tetrahedron: Asymmetry (2015), http://dx.doi.org/10.1016/j.tetasy.2015.11.001

K. Stanková et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
7
Table 3
corresponding oxazolidinone analogues 28, 29, were essentially
inactive (IC50 >100 M). Moreover, the rearranged product 15
Crystal data and structure refinement parameters for compound 15
l
demonstrated remarkable in vitro potency against Jurkat cells
comparable to that of cisplatin (Table 4). Likewise, 15 displayed
significant activity on an HCT-116 colon cancer cell line. Out of
15
Empirical formula
Formula weight
Temperature, T (K)
Wavelength, k (Å)
Crystal system
C25H29NO4S
439.55
173(2)
0.71073
Monoclinic
P21
the tested derivatives, homospisulosine
7 showed the best
inhibitory effects against all of the cell lines, with IC50 values com-
parable to or lower than those of traditional anticancer agents
(Table 4). On the other hand, sphingoid bases ent-7 and 10 dis-
played lower in vitro potency against Caco-2, MDA-MB-231,
HCT-116 and MCF-7 than homospisulosine 7, suggesting that the
(3S,4R)-amino alcohol moiety appears to be their determining fac-
tor for the biological activity. Interestingly, both compounds ent-7
and 10 had significantly higher potencies on leukaemia (Jurkat)
than on solid tumour cell lines. For the cyclic carbamates 33,
ent-33 and 36 as the protected form of the corresponding amino
alcohols 7, ent-7 and 10, respectively, the cytotoxicity was signifi-
cantly reduced. Similar results were also observed in the case of
compound (Z)-32.
Space group
Unit cell dimensions
a (Å)
b (Å)
10.6710(4)
8.2405(3) b = 96.347(4)°
13.5002(5)
1179.86(8)
2
1.237
0.167
468
c (Å)
V (Å3)
Formula per unit cell, Z
Dcalcd (g/cm3)
Absorption coefficient,
F(000)
Crystal size (mm)
h Range for data collection (°)
Index ranges
l
(mmꢀ1
)
0.7595 ꢂ 0.2112 ꢂ 0.1409
2.901–24.968
ꢀ12 6 h 6 12
ꢀ9 6 k 6 9
ꢀ16 6 l 6 15
4143 (0.0318)
Analytical
Independent reflections (Rint
Absorption correction
)
3. Conclusion
Max. and min. transmission
Refinement method
0.983 and 0.933
Full-matrix least-squares on F2
4143/1/283
In conclusion, we have accomplished the convergent total
synthesis of cytotoxic homospisulosine 7 and its 3-epi-analogue
Data/restraints/parameters
Goodness-of-fit on F2
1.042
10 from the commercially available and inexpensive
D-xylose.
Final R indices [I > 2
R indices (all data)
Flack x parameter
r
(I)]
R1 = 0.0358, wR2 = 0.0850
R1 = 0.0435, wR2 = 0.0899
ꢀ0.04(4)
The key transformations of our approach were the implementation
of an amino-bearing asymmetric centre via [3,3]-heterosigmat-
ropic rearrangements and the regioselective intramolecular
cyclization. In order to rationalize the stereochemical outcome of
the rearrangement processes, DFT calculations were carried out
and the obtained data were in excellent agreement with the exper-
imental results. A series of the prepared compounds was screened
for antiproliferative/cytotoxic activities against five cancer cell
lines. Among them, the final derivative 7 was the most promising
molecule with similar potency to etoposide and cisplatin, and
was taken up as a lead structure for further investigation. The
sphingoid bases ent-7 and 10 displayed significant activity only
on Jurkat cells. These obtained results also revealed that the
potency of the cytotoxicity is dependent on the stereochemistry
of the vicinal amino alcohol unit. The isothiocyanate 15 showed
Largest diff. peak and hole (e/Åꢀ3
)
0.291 and ꢀ0.305
MDA-MB-231 and MCF-7 (breast cancer cells), Caco-2 (human
colon carcinoma), HCT-116 (human colon carcinoma), Jurkat
(human acute T-lymphoblastic leukaemia), and a non-malignant
cell line NiH 3T3 (mouse fibroblasts) using MTT assay. The
obtained results are summarized in Table 4 as IC50 values. Com-
mercially available anticancer substances etoposide and cisplatin
were included as positive control33 in the case of cell lines Jurkat,
MCF-7 and MDA-MB-231 (Table 4), on HCT-116 and Caco-2 cells
they were not tested. For the sake of comparison, Table 4 also
includes IC50 values for ent-734 and ent-33,34 the preparation of
which will be reported in due course.
IC50 values less than 10 lM in the inhibition of the proliferation
As shown in Table 4, isothiocyanates 14 and 15 were found to
be more active than cyclic thiocarbamates 26, 27 as well as their
of the two cancer cell lines (Jurkat and HCT-116) and this com-
pound was further tested for cell cycle analysis. This study together
Table 4
Antiproliferative activities on five human cancer cell lines (MDA-MB-231, MCF-7, HCT-116, Caco-2 and Jurkat) and non-malignant mouse fibroblasts NiH 3T3
a
Compd no.
Cell line, IC50 SD (
HCT-116
l )
mol Lꢀ1
MDA-MB-231
MCF-7
Caco-2
Jurkat
NiH 3T3
14
15
26
27
42.2 4.2
29.3 5.1
>100
>100
>100
42.9 3.8
27.1 4.7
>100
>100
>100
32.4 3.7
<10
70.0
>100
>100
40.0 4.5
16.6 3.2
>100
>100
>100
33.8 6.2
<10
56.5 7.2
>100
38.7 2.9
33.3 4.4
76.8 6.7
91.1 5.9
>100
28
>100
29
>100
>100
>100
>100
>100
>100
(Z)-32
33
36
48.0 5.2
69.3 7.2
54.0 4.7
<10
50.0 4.3
56.9 5.6
34.2 3.6
<10
51.4 2.8
43.4 2.8
57.7 5.1
<10
45.5 3.9
62.5 3.7
65.3 2.9
<10
31.6 6.5
24.4 4.4
59.3 3.5
<10
42.3 4.2
41.9 6.3
49.3 2.8
<10
7
10
28.6 5.7
24.2 3.8
86.4 3.9
14.7 2.7
21.2 4.2
36.0 4.3
29.0 4.2
69.1 6.2
11.4 2.4
10.9 2.1
28.4 2.9
22.1 3.1
80.9 7.7
NT
28.9 4.1
28.6 5.3
86.2 7.3
NT
<10
<10
66.6 6.8
12 1.8
1.2 1.5
25.7 2.6
26.1 2.4
76.2 5.7
NT
ent-7b
ent-33b
Cisplatinc
Etoposidec
NT
NT
NT
NT-not tested.
a
The potency of compounds was determined using the MTT assay after 72 h incubation of cells and given as IC50 (concentration of a tested compound, which decreased the
amount of viable cells to 50% relative to untreated control cells, see Section 4.2).
b
Unpublished structure.34
c
Values for the clinically available anticancer drugs are reported in Ref. 33 and were utilized from the same source.
Please cite this article in press as: Stanková, K.; et al. Tetrahedron: Asymmetry (2015), http://dx.doi.org/10.1016/j.tetasy.2015.11.001

8
K. Stanková et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
with results of cytotoxic effect of the selected compounds on
human non-malignant endothelial cells HUVEC will be reported
in due course. Evaluation of the aforementioned structures by
employing a broader panel of cancer cell lines would be appropri-
ate to find selective compound as exemplified for derivatives 7,
ent-7, 10 and also 15.
(dd, 1H, J = 9.7 Hz, J = 5.6 Hz, H-7), 3.58–3.62 (m, 2H, H-5, H-7),
3.95–3.96 (m, 1H, H-6), 4.20 (q, 2H, J = 7.1 Hz, CH2), 4.48–4.51
(m, 3H, H-4, OCH2Ph), 4.57 (d, 1H, J = 11.3 Hz, OCH2Ph), 4.64 (d,
1H, J = 11.3 Hz, OCH2Ph), 6.14 (dd, 1H, J = 15.6 Hz, J = 1.9 Hz, H-2),
7.00 (dd, 1H, J = 15.6 Hz, J = 4.4 Hz, H-3), 7.26–7.37 (m, 10H, Ph);
13C NMR (100 MHz, CDCl3): d 14.2 (CH3), 60.4 (CH2), 70.7 (C-7),
70.8 (C-6), 71.1 (C-4), 73.5 (OCH2Ph), 74.8 (OCH2Ph), 80.7 (C-5),
121.7 (C-2), 127.9 (3 ꢂ CHPh), 128.1 (CHPh), 128.2 (2 ꢂ CHPh),
128.5 (4 ꢂ CHPh), 137.3 (Ci), 137.4 (Ci), 147.1 (C-3), 166.2 (C-1).
Anal. Calcd for C23H28O6: C, 68.98; H, 7.05. Found: C, 69.10; H, 6.97.
4. Experimental
4.1. Chemistry
All commercial reagents were used in the highest available
purity from Aldrich, Merck and Acros Organics without further
purification. Solvents were dried and purified before use according
to standard procedures. For flash column chromatography on silica
gel, Kieselgel 60 (0.040–0.063 mm, 230–400 mesh, Merck) was
used. Solvents for chromatography (n-hexane, ethyl acetate,
methanol, dichloromethane) were distilled before use. Thin layer
chromatography was run on Merck silica gel 60 F254 analytical
plates; detection was carried out with either ultraviolet light
(254 nm), or spraying with a solution of phosphomolybdic acid, a
basic potassium permanganate solution, or a solution of concen-
trated H2SO4, with subsequent heating. 1H NMR and 13C NMR spec-
tra were recorded in CDCl3, CD3OD and C6D6 on a Varian Mercury
Plus 400 FT NMR (400.13 MHz for 1H and 100.6 MHz for 13C) or on
4.1.2. Ethyl (4S,5R,6R,2Z)-5,7-bis(benzyloxy)-4,6-(isopropyliden-
edioxy)hept-2-enoate (Z)-23 and ethyl (4S,5R,6R,2E)-5,7-bis
(benzyloxy)-4,6-(isopropylidenedioxy)hept-2-enoate (E)-24
To a solution of the mixture of esters (E)-21/(Z)-22 (7.0 g,
17.5 mmol) in dry CH2Cl2 (96 mL) were successively added
2,2-DMP (6.5 mL, 52.42 mmol) and p-TsOH (0.13 g, 0.70 mmol).
After stirring for 3 h at room temperature, the mixture was diluted
with CH2Cl2 (460 mL) and washed with a saturated solution of
NaHCO3 (460 mL). The aqueous layer was then extracted with
another portion of CH2Cl2 (460 mL). The combined organic extracts
were dried over Na2SO4, the solvent was subsequently evaporated,
and the residue was chromatographed on silica gel (n-hexane/
ethyl acetate, 7:1). This procedure yielded 1.38 g (18%) of (Z)-23
26
and 5.39 g (70%) of (E)-24 as colourless oils. Ester (Z)-23: [
a]
=
D
a
Varian Premium COMPACT 600 (599.87 MHz for 1H and
+111.4 (c 0.28, CHCl3). IR (neat) mmax 3030, 2989, 2924, 2868,
150.84 MHz for 13C) spectrometer using TMS as internal reference.
For 1H, d are given in parts per million (ppm) relative to TMS
(d = 0.0), CD3OD (d = 4.84) and C6D6 (d = 7.15) and for 13C relative
to CDCl3 (d = 77.0), CD3OD (d = 49.05) and C6D6 (d = 128.02). The
multiplicity of the 13C NMR signals concerning the 13C-1H coupling
was determined by the DEPT method. Chemical shifts (in ppm) and
coupling constants (in Hz) were obtained by first-order analysis;
assignments were derived from COSY and H/C correlation spectra.
Infrared (IR) spectra were measured with a Nicolet 6700 FT-IR
spectrometer and expressed in v values (cmꢀ1). Optical rotations
were measured on a P-2000 Jasco polarimeter and reported as
1713, 1649, 1497, 1454, 1416, 1378, 1264, 1192, 1167, 1137,
1097, 1074, 1027, 939 cmꢀ1 1H NMR (400 MHz, CDCl3): d 1.29 (t,
;
3H, J = 7.1 Hz, CH3), 1.47 (s, 3H, CH3), 1.50 (s, 3H, CH3), 3.53 (dd,
1H, J = 9.3 Hz, J = 5.9 Hz, H-7), 3.56 (dd, 1H, J = 9.3 Hz, J = 7.1 Hz,
H-7), 3.70 (m, 1H, H-5), 4.15 (q, 2H, J = 7.1 Hz, CH2), 4.20–4.24
(m, 1H, H-6), 4.44–4.56 (m, 4H, 2 ꢂ OCH2Ph), 5.47–5.49 (m, 1H,
H-4), 5.69 (dd, 1H, J = 11.8 Hz, J = 1.4 Hz, H-2), 6.29 (dd, 1H,
J = 11.8 Hz, J = 7.1 Hz, H-3), 7.22–7.35 (m, 10H, Ph); 13C NMR
(100 MHz, CDCl3): d 14.2 (CH3), 19.3 (CH3), 29.7 (CH3), 60.3
(CH2), 69.4 (C-7), 70.0 (C-4), 71.2 (C-6), 71.8 (C-5), 73.4 (OCH2Ph),
74.5 (OCH2Ph), 98.9 (Cq), 120.0 (C-2), 127.7 (2 ꢂ CHPh), 127.9
(2 ꢂ CHPh), 128.1 (2 ꢂ CHPh), 128.3 (2 ꢂ CHPh), 128.4 (2 ꢂ CHPh),
138.0 (Ci), 138.1 (Ci), 147.8 (C-3), 165.7 (C-1). Anal. Calcd for
follows: [a]D (c in grams per 100 mL, solvent). Melting points were
recorded on a Kofler hot block, and are uncorrected. Microwave
reactions were carried out on the focused microwave system
(CEM Discover). The temperature content of the vessel was moni-
tored using a calibrated infrared sensor mounted under the vessel.
At the end of all reactions, the contents of vessel were cooled
rapidly using a stream of compressed air. Small quantities of
C
24: [
26H32O6: C, 70.89; H, 7.32. Found: C, 70.93; H, 7.38. Ester (E)-
a]
D
24 = ꢀ22.7 (c 0.22, CHCl3). IR (neat) mmax 2987, 2870, 1714,
1661, 1496, 1454, 1381, 1303, 1258, 1201, 1159, 1094, 1043,
981 cmꢀ1 1H NMR (400 MHz, CDCl3): d 1.27 (t, 3H, J = 7.1 Hz,
;
CH3), 1.46 (s, 3H, CH3), 1.49 (s, 3H, CH3), 3.45–3.46 (m, 1H, H-5),
3.53 (dd, 1H, J = 9.1 Hz, J = 5.5 Hz, H-7), 3.63 (dd, 1H, J = 9.1 Hz,
J = 7.6 Hz, H-7), 4.13–4.21 (m, 3H, H-6, CH2), 4.45 (d, 1H,
J = 11.8 Hz, OCH2Ph), 4.50–4.57 (m, 4H, H-4, OCH2Ph), 6.13 (dd,
1H, J = 15.7 Hz, J = 1.8 Hz, H-2), 6.87 (dd, 1H, J = 15.7 Hz,
J = 4.5 Hz, H-3), 7.22–7.36 (m, 10H, Ph); 13C NMR (100 MHz,
CDCl3): d 14.2 (CH3), 19.0 (CH3), 29.5 (CH3), 60.3 (CH2), 69.1
(C-7), 71.4 (C-5, C-6), 72.0 (C-4), 73.5 (OCH2Ph), 74.2 (OCH2Ph),
99.0 (Cq), 122.0 (C-2), 127.7 (CHPh), 127.8 (CHPh), 127.9 (2 ꢂ CHPh),
128.2 (2 ꢂ CHPh), 128.4 (4 ꢂ CHPh), 137.7 (2 ꢂ Ci), 144.4 (C-3),
166.1 (C-1). Anal. Calcd for C26H32O6: C, 70.89; H, 7.32. Found: C,
70.91; H, 7.40.
reagents
(lL) were measured with appropriate syringes
(Hamilton). All reactions were performed under an atmosphere
of nitrogen, unless otherwise noted.
4.1.1. Ethyl (4S,5R,6R,2E)-5,7-bis(benzyloxy)-4,6-dihydroxyhept-
2-enoate (E)-21 and ethyl (4S,5R,6R,2Z)-5,7-bis(benzyloxy)-4,6-
dihydroxyhept-2-enoate (Z)-22
To a solution of the known 3,5-di-O-benzyl-D
-xylofuranose 2026
(14.0 g, 42.37 mmol) in dry CH2Cl2 (285 mL) were successively
added the stabilized ylide (Ph3P@CHCO2Et, 26.57 g, 76.27 mmol)
and catalytic amounts of benzoic acid (0.513 g, 4.2 mmol). After
stirring of the mixture for 5 h at room temperature, the solvent
was evaporated, and the residue was chromatographed on silica
gel (n-hexane/ethyl acetate, 3:1) to afford 15.3 g (90%) of a mixture
4.1.3. (4S,5R,6R,2E)-5,7-Bis(benzyloxy)-4,6-(isopropylidenedioxy)
hept-2-en-1-ol 2528
of
a,b-unsaturated esters (E)-21 and (Z)-22. Repeated chromatog-
To a solution of ester (E)-24 (5.3 g, 12.03 mmol) in CH2Cl2
(55 mL), which had been pre-cooled to –15 °C, was added
diisobutylaluminum hydride (36.1 mL, 43.3 mmol, ꢁ1.2 M toluene
solution) dropwise, and the resulting mixture was stirred for
15 min at ꢀ15 °C. The excess hydride was decomposed by the
addition of MeOH (8.5 mL), and after warming to room tempera-
ture, the mixture was poured into a 30% solution of K/Na tartrate
(180 mL). After stirring for 1.5 h, the aqueous phase was extracted
raphy on silica gel (n-hexane/ethyl acetate, 3:1) gave only an
analytical sample of the major geometrical isomer (E)-21 as a
colourless oil.
Ester (E)-21: [
a
]
D
23 = ꢀ9.4 (c 0.18, CHCl3). IR (neat) mmax 3450,
3030, 2869, 1779, 1712, 1495, 1451, 1375, 1184, 1074, 1025,
901 cmꢀ1
;
1H NMR (400 MHz, CDCl3): d 1.29 (t, 3H, J = 7.1 Hz,
CH3), 2.66 (m, 1H, OH), 3.06 (br d, 1H, J = 5.9 Hz, OH), 3.52
Please cite this article in press as: Stanková, K.; et al. Tetrahedron: Asymmetry (2015), http://dx.doi.org/10.1016/j.tetasy.2015.11.001

K. Stanková et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
9
with CH2Cl2 (2 ꢂ 250 mL). The combined organic layers were dried
over Na2SO4, after which the solvent was evaporated, and the resi-
due was purified by flash chromatography on silica gel (n-hexane/
ethyl acetate, 2:1) to give 4.7 g (98%) of alcohol 2528 as a colourless
the reaction times and temperatures, see Table 1). After cooling to
room temperature, the solvent was evaporated, and the crude mix-
ture was chromatographed on silica gel (n-hexane/ethyl acetate,
20:1) to give the corresponding rearranged products 14 and 15
(for the combined yields, see Table 1). In order to obtain greater
amounts of pure isothiocyanates 14 and 15, they could be prepared
on a multi-gram scale by the conventional method in n-heptane at
90 °C.
oil; [
(neat)
1199, 1168, 1095, 1064, 1026 cmꢀ1
a
]
m
25 = +4.2 (c 1.28, CHCl3); lit.28
[
a
]
24 = ꢀ38.8 (c 1.24, CHCl3). IR
D
D
max 3426, 3030, 2990, 2865, 1735, 1496, 1454, 1380, 1262,
;
1H NMR (400 MHz, CDCl3): d
1.47 (s, 6H, 2 ꢂ CH3), 3.35–3.36 (m, 1H, H-5), 3.54 (dd, 1H,
J = 9.0 Hz, J = 5.4 Hz, H-7), 3.66 (dd, 1H, J = 9.0 Hz, J = 7.8 Hz, H-7),
3.98 (dd, 1H, J = 13.8 Hz, J = 5.5 Hz, H-1), 4.02 (dd, 1H, J = 13.8 Hz,
J = 5.3 Hz, H-1), 4.13–4.16 (m, 1H, H-6), 4.34 (m, 1H, H-4), 4.47
(d, 1H, J = 11.8 Hz, OCH2Ph), 4.53 (d, 1H, J = 11.7 Hz, OCH2Ph),
4.54 (d, 1H, J = 11.9 Hz, OCH2Ph), 4.63 (d, 1H, J = 11.9 Hz, OCH2Ph),
5.68 (ddt, 1H, J = 15.6 Hz, J = 6.5 Hz, J = 1.4 Hz, J = 1.4 Hz, H-3), 5.86
(dtd, 1H, J = 15.6 Hz, J = 5.3 Hz, J = 5.3 Hz, J = 0.9 Hz, H-2), 7.25–7.36
(m, 10H, Ph); 13C NMR (100 MHz, CDCl3): d 19.1 (CH3), 29.6 (CH3),
62.9 (C-1), 69.1 (C-7), 71.2 (C-6), 72.4 (C-5), 73.1 (C-4), 73.5 (OCH2-
Ph), 74.3 (OCH2Ph), 98.9 (Cq), 127.6 (CHPh), 127.8 (CHPh), 127.9
(2 ꢂ CHPh), 128.1 (2 ꢂ CHPh), 128.4 (2 ꢂ CHPh), 128.6 (2 ꢂ CHPh),
128.7 (C-3), 131.6 (C-2), 137.8 (Ci), 138.3 (Ci). Anal. Calcd for
4.1.5.2. Microwave-assisted synthesis.
Thiocyanate 18
(0.10 g, 0.227 mmol) was weighed into a 10-mL glass pressure
microwave tube equipped with a magnetic stir bar. n-Heptane
(1.9 mL) was then added, the tube was closed with a silicon septum
and the resulting solution was subjected to microwave irradiation
(for the temperatures and reaction times, see Table 1). Removal of
the solvent and chromatography of the residue on silica gel
(n-hexane/ethyl acetate, 20:1) afforded isothiocyanates 14 and
15 (for the combined yields, see Table 1). Diastereoisomer 14:
colourless oil; [
2915, 2868, 2087, 1496, 1454, 1409, 1381, 1358, 1310, 1264,
1201, 1168, 1085, 1045, 1027 cmꢀ1 1H NMR (400 MHz, CDCl3): d
a]
25 = +52.7 (c 0.70, CHCl3). IR (neat) mmax 2991,
D
C24H30O5: C, 72.34; H, 7.59. Found: C, 72.43; H, 7.62.
;
1.39 (s, 3H, CH3), 1.40 (s, 3H, CH3), 3.53 (dd, 1H, J = 9.1 Hz,
J = 5.5 Hz, H-100), 3.63–3.68 (m, 2H, H-5, H-100), 3.73 (dd, 1H,
J = 9.4 Hz, J = 1.6 Hz, H-6), 4.10 (ddd, 1H, J = 7.8 Hz, J = 5.5 Hz,
J = 1.5 Hz, H-4), 4.45–4.50 (m, 2H, H-10, OCH2Ph), 4.53 (d, 1H,
J = 11.8 Hz, OCH2Ph), 4.67 (d, 1H, J = 11.6 Hz, OCH2Ph), 4.71
(d, 1H, J = 11.6 Hz, OCH2Ph), 5.28 (ddd, 1H, J = 10.4 Hz, J = 1.5 Hz,
J = 0.6 Hz, H-30cis), 5.43 (ddd, 1H, J = 17.0 Hz, J = 1.7 Hz, J = 0.6 Hz,
H-30trans), 5.97 (ddd, 1H, J = 17.0 Hz, J = 10.4 Hz, J = 4.7 Hz, H-20),
7.27–7.37 (m, 10H, Ph); 13C NMR (100 MHz, CDCl3): d 19.1 (CH3),
29.3 (CH3), 58.4 (C-10), 68.9 (C-100), 70.0 (C-5), 71.3 (C-4), 73.6
(OCH2Ph), 74.9 (OCH2Ph, C-6), 99.3 (Cq), 116.9 (C-30), 127.8
(3 ꢂ CHPh), 127.9 (CHPh), 128.0 (2 ꢂ CHPh), 128.3 (2 ꢂ CHPh),
128.5 (2 ꢂ CHPh), 132.9 (C-20), 134.3 (NCS), 137.6 (Ci), 138.0 (Ci).
Anal. Calcd for C25H29NO4S: C, 68.31; H, 6.65; N, 3.19. Found: C,
68.33; H, 6.69; N, 3.24. Diastereoisomer 15: white crystals, mp
4.1.4. (4R,5R,6S)-5-(Benzyloxy)-4-[(benzyloxy)methyl]-2,2-
dimethyl-6-[(E)-30-thiocyanatoprop-10-en-10-yl)]-1,3-dioxane 18
To a solution of the known alcohol 2528 (3.7 g, 9.29 mmol) in
dry CH2Cl2 (80 mL), which had been pre-cooled to 0 °C, were suc-
cessively added Et3N (2.61 mL, 18.6 mmol) and MsCl (1.44 mL,
18.6 mmol). The resulting mixture was stirred for 15 min at 0 °C
and then at room temperature for another 20 min. After evapora-
tion of the solvent, the residue was diluted with Et2O, the salts
were filtered off, washed with Et2O, and the solvent was evapo-
rated. The obtained crude product was used in the next reaction
without purification.
To a solution of the crude mesylate (4.42 g, 9.29 mmol) in dry
acetonitrile (80 mL), which had been pre-cooled to 5 °C, was added
KSCN (1.35 g, 13.94 mmol), and the resulting mixture was then
stirred at room temperature for 22 h before evaporating off the sol-
vent. To the obtained residue, Et2O was added to give salts, which
were removed by filtration. The filtrate was concentrated to pro-
vide a pale yellow oil, which was subjected to flash chromatogra-
phy on silica gel (n-hexane/ethyl acetate, 5:1) to yield 3.51 g
(86%) of compound 18 as white crystals; mp 48–49 °C (recrystal-
78–79 °C (recrystallized from n-hexane);
CHCl3). IR (neat) mmax 3029, 2991, 2916, 2868, 2043, 1496, 1454,
1381, 1266, 1201, 1171, 1156, 1095, 1043, 1027 cmꢀ1 1H NMR
[a]
25 = +2.9 (c 0.24,
D
;
(400 MHz, CDCl3): d 1.48 (s, 3H, CH3), 1.50 (s, 3H, CH3), 3.45
(m, 1H, H-5), 3.55 (dd, 1H, J = 9.1 Hz, J = 5.3 Hz, H-100), 3.67–3.72
(m, 1H, H-100), 3.77 (dd, 1H, J = 9.0 Hz, J = 1.4 Hz, H-6), 4.06–4.10
(m, 1H, H-4), 4.48–4.58 (m, 4H, H-10, OCH2Ph), 4.73 (d, 1H,
J = 11.9 Hz, OCH2Ph), 5.22–5.25 (m, 1H, H-30), 5.33–5.38 (m, 1H,
H-30), 5.42–5.50 (m, 1H, H-20), 7.23–7.37 (m, 10H, Ph); 13C NMR
(100 MHz, CDCl3): d 18.9 (CH3), 29.4 (CH3), 60.6 (C-10), 68.6
(C-100), 69.5 (C-5), 71.6 (C-4), 73.7 (2 ꢂ OCH2Ph), 75.5 (C-6), 99.7
(Cq), 119.8 (C-30), 127.4 (2 ꢂ CHPh), 127.7 (CHPh), 128.0 (CHPh),
128.1 (2 ꢂ CHPh), 128.4 (2 ꢂ CHPh), 128.5 (2 ꢂ CHPh), 130.5 (C-20),
136.0 (NCS), 137.5 (Ci), 138.1 (Ci). Anal. Calcd for C25H29NO4S: C,
68.31; H, 6.65; N, 3.19. Found: C, 68.25; H, 6.70; N, 3.14.
lized from n-heptane); [a]
25 = +15.0 (c 0.22, CHCl3). IR (neat) mmax
D
3060, 3030, 2984, 2912, 2866, 2147, 1496, 1472, 1454, 1406,
1379, 1343, 1255, 1199, 1178, 1139, 1094, 1080, 1044,
1027 cmꢀ1
;
1H NMR (400 MHz, CDCl3): d 1.47 (s, 6H, 2 ꢂ CH3),
3.40–3.49 (m, 3H, 2 ꢂ H-30, H-5), 3.53 (dd, 1H, J = 9.1 Hz,
J = 5.5 Hz, H-100), 3.61–3.65 (m, 1H, H-100), 4.12–4.15 (m, 1H, H-4),
4.39–4.41 (m, 1H, H-6), 4.45 (d, 1H, J = 11.8 Hz, OCH2Ph), 4.51 (d,
1H, J = 11.8 Hz, OCH2Ph), 4.59 (d, 1H, J = 12.0 Hz, OCH2Ph), 4.62
(d, 1H, J = 12.0 Hz, OCH2Ph), 5.73 (dd, 1H, J = 15.4 Hz, J = 5.7 Hz,
H-10), 5.82–5.90 (m, 1H, H-20), 7.26–7.35 (m, 10H, Ph); 13C NMR
(100 MHz, CDCl3): d 19.1 (CH3), 29.6 (CH3), 35.8 (C-30), 69.1
(C-100), 71.2 (C-4), 72.1 (C-5), 72.5 (C-6), 73.5 (OCH2Ph), 74.4
(OCH2Ph), 99.0 (Cq), 111.9 (SCN), 124.6 (C-20), 127.7 (CHPh), 127.8
(CHPh), 127.9 (2 ꢂ CHPh), 128.2 (2 ꢂ CHPh), 128.4 (4 ꢂ CHPh),
134.5 (C-10), 137.8 (Ci), 138.2 (Ci). Anal. Calcd for C25H29NO4S: C,
68.31; H, 6.65; N, 3.19. Found: C, 68.25; H, 6.71; N, 3.09.
4.1.6. N-{(10S)-10-[(400S,500R,600R)-500-(Benzyloxy)-600-[(benzyloxy)
methyl]-200,200-dimethyl-100,300-dioxan-400-yl]allyl}-2,2,2-trichloro-
acetamide 16 and N-{(10R)-10-[(400S,500R,600R)-500-(benzyloxy)-600-
[(benzyloxy)methyl]-200,200-dimethyl-100,300-dioxan-400-yl]allyl}-2,
2,2-trichloroacetamide 17
To a solution of alcohol 2528 (80 mg, 0.2 mmol) in dry THF
(0.65 mL), which had been pre-cooled to 0 °C, was added NaH
(10.7 mg, 0.445 mmol, 60% dispersion in mineral oil). The resulting
4.1.5. (4R,5R,6S)-5-(Benzyloxy)-4-[(benzyloxy)methyl]-6-[(10S)-
10-isothiocyanatoallyl)]-2,2-dimethyl-1,3-dioxane 14 and (4R,
5R,6S)-5-(benzyloxy)-4-[(benzyloxy)methyl]-6-[(10R)-10-isothio-
cyanatoallyl)]-2,2-dimethyl-1,3-dioxane 15
suspension was stirred at 0 °C for 5 min, then CCl3CN (24 lL,
0.24 mmol) was added dropwise. After stirring at 0 °C for another
15 min, the mixture was filtered through a small pad of Celite,
the solvent was evaporated, and the obtained crude trichloroace-
timidate 19 was used immediately in the subsequent rearrange-
ments without further purification.
4.1.5.1. Conventional method.
Thiocyanate 18 (0.10 g,
0.227 mmol) was dissolved in n-heptane (1.9 mL) and the resulting
solution was stirred and heated under a nitrogen atmosphere (for
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10
K. Stanková et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
4.1.6.1. Microwave-assisted synthesis.
Imidate 19 (108 mg,
2 ꢂ H-200), 5.80 (ddd, 1H, J = 17.3 Hz, J = 10.1 Hz, J = 8.2 Hz, H-100),
0.2 mmol) was weighed into a 10-mL glass pressure microwave
tube equipped with a magnetic stirrer bar. o-Xylene (2.7 mL) and
anhydrous K2CO3 (30 mg, 0.22 mmol) were then added. The tube
was closed with a silicon septum and the reaction mixture was
subjected to microwave irradiation (for the temperatures and reac-
tion times, see Table 2). Removal of the solvent and chromatogra-
phy of the residue on silica gel (n-hexane/ethyl acetate, 5:1) gave
the corresponding trichloroacetamides 16 and 17 as white crystals
(for the combined yields, see Table 2).
7.26–7.36 (m, 10H, Ph), 7.80 (s, 1H, NH); 13C NMR (100 MHz,
CDCl3): d 60.5 (C-4), 69.0 (C-20), 70.6 (C-30), 73.3 (OCH2Ph), 74.3
(OCH2Ph), 75.7 (C-10), 85.6 (C-5), 121.7 (C-200), 127.7 (2 ꢂ CHPh),
127.8 (CHPh), 127.9 (CHPh), 128.2 (2 ꢂ CHPh), 128.4 (4ꢂ CHPh),
131.0 (C-100), 137.4 (Ci), 137.6 (Ci), 189.3 (C-2). Anal. Calcd for
C22H25NO4S: C, 66.14; H, 6.31; N, 3.51. Found: C, 66.35; H, 6.41;
N, 3.60.
4.1.8. (4R,5S)-5-[(10S,20R)-10,30-Bis(benzyloxy)-20-hydroxypropyl]-
4-vinyloxazolidine-2-thione 27
Using the same procedure as described for the preparation of
26, isothiocyanate 15 (0.75 g, 1.71 mmol) and p-TsOH (33 mg,
0.17 mmol) furnished, after stirring (6 h) and flash chromatogra-
To obtain a greater amount of the rearranged products 16 and
17, the aforementioned procedure was repeated several times at
130 °C, using 0.25 g of the starting imidate 19. Diastereoisomer
16: mp 82–83 °C (recrystallized from n-hexane/ethyl acetate);
[
a
]
24 = ꢀ47.1 (c 0.14, CHCl3). IR (neat) mmax 3344, 2991, 2917,
phy through a small pad of silica gel (n-hexane/ethyl acetate,
D
23
2870, 1704, 1498, 1455, 1381, 1366, 1258, 1202, 1166, 1093,
3:1), 0.66 g (97%) of compound 27 as a colourless oil; [
a
]
=
D
1027 cmꢀ1
;
1H NMR (400 MHz, CDCl3): d 1.41 (s, 3H, CH3), 1.45 (s,
+67.8 (c 0.37, CHCl3). IR (neat) mmax 3214, 3028, 2867, 1730,
3H, CH3), 3.54 (dd, 1H, J = 9.0 Hz, J = 5.3 Hz, H-1000), 3.65 (m, 1H, H-
500), 3.72 (t, 1H, J = 8.9 Hz, H-1000), 4.04 (dd, 1H, J = 5.0 Hz, J = 1.7 Hz,
H-400), 4.10–4.15 (m, 1H, H-600), 4.47–4.53 (m, 3H, OCH2Ph), 4.67–
4.74 (m, 2H, H-10, OCH2Ph), 5.33–5.38 (m, 2H, 2 ꢂ H-30), 5.77
(ddd, 1H, J = 17.2 Hz, J = 10.4 Hz, J = 4.5 Hz, H-20), 7.27–7.32
(m, 10H, Ph), 8.21 (br d, J = 7.2 Hz, NH); 13C NMR (100 MHz, CDCl3):
d 19.0 (CH3), 29.3 (CH3), 56.2 (C-10), 68.3 (C-1000), 71.3 (C-600), 71.5 (C-
400 or C-500), 71.6 (C-400 or C-500), 73.6 (OCH2Ph), 74.2 (OCH2Ph), 91.8
(C-2), 99.0 (Cq), 117.7 (C-30), 128.0 (CHPh), 128.1 (5 ꢂ CHPh), 128.5
(4 ꢂ CHPh), 132.3 (C-20), 137.2 (Ci), 137.3 (Ci), 162.4 (C-1). Anal.
Calcd for C26H30Cl3NO5: C, 57.52; H, 5.57; N, 2.58. Found: C,
57.77; H, 5.82; N, 2.71. Diastereoisomer 17: mp 69–70 °C (recrystal-
1495, 1453, 1370, 1240, 1179, 1070, 1027, 987, 927 cmꢀ1 1H
;
NMR (400 MHz, CDCl3): d 2.65 (br d, 1H, J = 5.6 Hz, OH), 3.53 (dd,
1H, J = 9.6 Hz, J = 5.5 Hz, H-30), 3.58 (dd, 1H, J = 9.6 Hz, J = 6.2 Hz,
H-30), 3.72 (dd, 1H, J = 5.2 Hz, J = 3.5 Hz, H-10), 3.99 (m, 1H, H-20),
4.28–4.32 (m, 1H, H-4), 4.49 (m, 2H, OCH2Ph), 4.66–4.71 (m, 2H,
H-5, OCH2Ph), 4.75 (d, 1H, J = 11.5 Hz, OCH2Ph), 5.15 (d, 1H,
J = 17.0 Hz, H-200trans), 5.22 (d, 1H, J = 10.1 Hz, H-200cis), 5.70 (ddd,
1H, J = 17.4 Hz, J = 10.1 Hz, J = 7.6 Hz, H-100), 7.29–7.37 (m, 10H,
Ph), 7.66 (s, 1H, NH); 13C NMR (100 MHz, CDCl3): d 60.7 (C-4),
69.0 (C-20), 70.8 (C-30), 73.5 (OCH2Ph), 74.4 (OCH2Ph), 76.8 (C-10),
87.3 (C-5), 120.0 (C-200), 128.0 (3 ꢂ CHPh), 128.2 (CHPh), 128.5
(4 ꢂ CHPh), 128.6 (2 ꢂ CHPh), 134.0 (C-100), 137.2 (Ci), 137.6 (Ci),
188.8 (C-2). Anal. Calcd for C22H25NO4S: C, 66.14; H, 6.31; N,
3.51. Found: C, 66.22; H, 6.45; N, 3.59.
lized from n-hexane/CH2Cl2); [
a
]
24 = ꢀ28.3 (c 0.12, CHCl3). IR (neat)
D
mmax 3416, 2991, 2917, 2869, 1767, 1709, 1497, 1454, 1381, 1245,
1202, 1170, 1095, 1046, 1028 cmꢀ1 1H NMR (400 MHz, CDCl3): d
;
1.44 (s, 3H, CH3), 1.46 (s, 3H, CH3), 3.47–3.51 (m, 2H, H-500, H-1000),
3.56–3.60 (m, 1H, H-1000), 3.97 (dd, 1H, J = 4.6 Hz, J = 1.6 Hz, H-400),
4.07–4.10 (m, 1H, H-600), 4.39–4.42 (m, 2H, H-10, OCH2Ph), 4.46 (d,
1H, J = 11.7 Hz, OCH2Ph), 4.57 (d, 1H, J = 11.7 Hz, OCH2Ph), 4.64
(d, 1H, J = 11.7 Hz, OCH2Ph), 5.24 (d, 1H, J = 10.3 Hz, H-30cis), 5.29
(d, 1H, J = 17.2 Hz, H-30trans), 5.78 (ddd, 1H, J = 17.2 Hz, J = 10.3 Hz,
J = 6.9 Hz, H-20), 7.25–7.35 (m, 10H, Ph), 7.47 (br d, 1H, J = 5.8 Hz,
NH); 13C NMR (100 MHz, CDCl3): d 19.0 (CH3), 29.4 (CH3), 55.4
(C-10), 68.7 (C-1000), 71.3 (C-500), 71.7 (C-600), 72.6 (C-400), 73.5
(OCH2Ph), 74.1 (OCH2Ph), 92.7 (C-2), 99.4 (Cq), 118.5 (C-30), 127.7
(CHPh), 127.8 (2 ꢂ CHPh), 127.9 (3 ꢂ CHPh), 128.3 (2 ꢂ CHPh), 128.4
(2 ꢂ CHPh), 133.5 (C-20), 137.6 (Ci), 137.8 (Ci), 161.0 (C-1). Anal.
Calcd for C26H30Cl3NO5: C, 57.52; H, 5.57; N, 2.58. Found: C,
57.63; H, 5.77; N, 2.69.
4.1.9. (4S,5S)-5-[(10S,20R)-10,30-Bis(benzyloxy)-20-hydroxypropyl]-
4-vinyloxazolidin-2-one 28
4.1.9.1. Modification of oxazolidinone-2-thione 26 into
28.
Mesitylnitrile oxide (0.77 g, 4.78 mmol) was added to a
solution of 26 (1.9 g, 4.76 mmol) in dry acetonitrile (46 mL). The
mixture was stirred for 1 h at room temperature before the solvent
was evaporated. The residue was chromatographed on silica gel (n-
hexane/ethyl acetate, 1:1) to afford 1.79 g (98%) of derivative 28 as
white crystals.
4.1.9.2. Modification of diol 30 into 28.
At first, DBU
(0.08 mL, 0.54 mmol) was added to a solution of 30 (0.68 g,
1.35 mmol) in dry CH2Cl2 (16 mL), which had been pre-cooled to
0 °C. The resulting mixture was stirred at 0 °C for 10 min and then
at room temperature for 48 h, before evaporating off the solvent.
The residue was subjected to flash chromatography through a
short silica gel column (n-hexane/ethyl acetate, 1:1) to give
0.446 g (86%) of compound 28. Oxazolidinone 28: mp 70–71 °C
4.1.7. (4S,5S)-5-[(10S,20R)-10,30-Bis(benzyloxy)-20-hydroxypropyl]-
4-vinyloxazolidine-2-thione 26
To a solution of the major isothiocyanate 14 (2.25 g, 5.12 mmol)
in MeOH (100 mL) was added p-TsOH (97 mg, 0.51 mmol), and the
resulting mixture was stirred at room temperature. After 8 h, no
starting material was identified in the reaction mixture (judged
by TLC). The solvent was evaporated, and the residue was flash-
chromatographed through a short silica gel column (n-hexane/
ethyl acetate, 1:1) to give 1.9 g (93%) of white crystalline derivative
26; mp 104–105 °C (recrystallized from n-hexane/ethyl acetate);
(recrystallized from n-hexane/ethyl acetate);
[
a
]
27 = ꢀ26.5 (c
D
0.26, CHCl3). IR (neat) mmax 3278, 3030, 2864, 2359, 2341, 1736,
1496, 1453, 1387, 1322, 1217, 1070, 1027 cmꢀ1
;
1H NMR
(400 MHz, CDCl3): d 2.52 (d, 1H, J = 8.0 Hz, OH), 3.46 (dd, 1H,
J = 9.4 Hz, J = 6.1 Hz, H-30), 3.53 (dd, 1H, J = 9.4 Hz, J = 6.7 Hz, H-
30), 3.75 (dd, 1H, J = 7.6 Hz, J = 2.2 Hz, H-10), 3.89 (ddd, 1H,
J = 6.6 Hz, J = 6.2 Hz, J = 2.2 Hz, H-20), 4.26–4.30 (m, 1H, H-4), 4.41
(d, 1H, J = 11.8 Hz, OCH2Ph), 4.49 (d, 1H, J = 11.8 Hz, OCH2Ph),
4.54 (d, 1H, J = 11.1 Hz, OCH2Ph), 4.83 (d, 1H, J = 11.1 Hz, OCH2Ph),
4.95–5.00 (m, 1H, H-5), 5.30 (d, 1H, J = 10.1 Hz, H-200cis), 5.35 (d, 1H,
J = 17.2 Hz, H-200trans), 5.60 (s, 1H, NH), 5.86 (ddd, 1H, J = 17.2 Hz,
J = 10.1 Hz, J = 8.2 Hz, H-100), 7.26–7.37 (m, 10H, Ph); 13C NMR
(100 MHz, CDCl3): d 57.3 (C-4), 68.9 (C-20), 70.8 (C-30), 73.3 (OCH2-
Ph), 74.2 (OCH2Ph), 76.0 (C-10), 80.1 (C-5), 120.5 (C-200), 127.7
(2 ꢂ CHPh), 127.8 (CHPh), 127.9 (CHPh), 128.1 (2 ꢂ CHPh), 128.4
(4 ꢂ CHPh), 132.9 (C-100), 137.6 (Ci), 137.7 (Ci), 159.0 (C-2). Anal.
[
a
]
27 = ꢀ63.8 (c 0.34, CHCl3). IR (neat) mmax 3327, 3192, 3023,
D
2942, 2857, 2359, 2341, 1734, 1497, 1452, 1401, 1377, 1362,
1338, 1320, 1277, 1259, 1131, 1085, 1062, 1038, 1022 cmꢀ1 1H
;
NMR (400 MHz, CDCl3): d 2.69 (d, 1H, J = 7.7 Hz, OH), 3.46 (dd,
1H, J = 9.5 Hz, J = 6.0 Hz, H-30), 3.54 (dd, 1H, J = 9.5 Hz, J = 6.7 Hz,
H-30), 3.79 (dd, 1H, J = 7.6 Hz, J = 2.3 Hz, H-10), 3.86–3.90 (m, 1H,
H-20), 4.35–4.42 (m, 2H, H-4, OCH2Ph), 4.47 (d, 1H, J = 11.8 Hz,
OCH2Ph), 4.56 (d, 1H, J = 11.1 Hz, OCH2Ph), 4.85 (d, 1H,
J = 11.1 Hz, OCH2Ph), 5.10–5.14 (m, 1H, H-5), 5.31–5.38 (m, 2H,
Please cite this article in press as: Stanková, K.; et al. Tetrahedron: Asymmetry (2015), http://dx.doi.org/10.1016/j.tetasy.2015.11.001

K. Stanková et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
11
Calcd for C22H25NO5: C, 68.91; H, 6.57; N, 3.65. Found: C, 68.91; H,
6.57; N, 3.65.
2865, 1705, 1644, 1497, 1454, 1408, 1244, 1209, 1088,
1027 cmꢀ1 1H NMR (400 MHz, CDCl3): d 2.49 (d, 1H, J = 6.6 Hz,
;
OH), 2.77 (d, 1H, J = 3.6 Hz, OH), 3.53 (dd, 1H, J = 9.6 Hz,
J = 5.6 Hz, H-70), 3.56–3.62 (m, 2H, H-70, H-50), 3.95–4.04 (m, 2H,
H-60, H-40), 4.49–4.60 (m, 4H, H-30, OCH2Ph), 4.67 (d, 1H,
J = 11.3 Hz, OCH2Ph), 5.25–5.30 (m, 2H, 2 ꢂ H-10), 5.83 (ddd, 1H,
J = 17.1 Hz, J = 10.4 Hz, J = 5.7 Hz, H-20), 7.21 (br d, 1H, J = 8.2 Hz,
NH), 7.26–7.37 (m, 10H, Ph); 13C NMR (100 MHz, CDCl3): d 54.5
(C-30), 69.7 (C-60), 70.6 (C-70), 71.9 (C-40), 73.4 (OCH2Ph), 74.9
(OCH2Ph), 78.6 (C-50), 92.7 (C-2), 117.7 (C-10), 127.9 (3 ꢂ CHPh),
128.3 (2 ꢂ CHPh), 128.4 (CHPh), 128.5 (2 ꢂ CHPh), 128.7 (2 ꢂ CHPh),
132.3 (C-20), 137.2 (Ci), 137.5 (Ci), 161.6 (C-1). Anal. Calcd for
4.1.10. (4R,5S)-5-[(10S,20R)-10,30-Bis(benzyloxy)-20-hydroxypro-
pyl]-4-vinyloxazolidin-2-one 29
4.1.10.1. Modification of oxazolidinone-2-thione 27 into
29.
According to the same reaction conditions as described
for the preparation of 28, compound 27 (0.66 g, 1.65 mmol) and
mesitylnitrile oxide (0.265 g, 1.64 mmol) afforded, after stirring
(1 h) and chromatography on silica gel (n-hexane/ethyl acetate,
1:1), 0.60 g (94%) of crystalline derivative 29.
4.1.10.2. Modification of diol 31 into 29.
procedure as described for the preparation of 28, compound 31
(0.51 g, 1.01 mmol) and DBU (30 L, 0.202 mmol) provided, after
Using the same
C23H26Cl3NO5: C, 54.94; H, 5.21; N, 2.79. Found: C, 55.45; H,
5.39; N, 2.85.
l
stirring (17 h) and chromatography on silica gel (n-hexane/ethyl
4.1.13. (4S,5S)-4-Ethyl-5-[(10S,20R)-10,20,30-trihydroxypropyl]
oxazolidin-2-one 11
acetate, 1:1), 0.37 g (95%) of carbamate 29. Oxazolidinone 29: mp
23
96–97 °C (recrystallized from n-hexane/ethyl acetate); [
a]
=
A solution of 28 (0.50 g, 1.30 mmol) in EtOH (10.5 mL) was
treated with 20% Pd(OH)2/C (46 mg) and 10% Pd/C (92 mg). The
resulting mixture was stirred at room temperature under an atmo-
sphere of hydrogen for 1 h. Next, the catalyst was removed by filtra-
tion through a pad of Celite, the filtrate was concentrated, and the
residue was chromatographed on silica gel (ethyl acetate/MeOH,
10:1) to afford 0.21 g (79%) of triol 11 as white crystals; mp 157–
D
+36.2 (c 0.26, CHCl3). IR (neat) mmax 3228, 2922, 2859, 1747,
1731, 1496, 1452, 1403, 1361, 1304, 1245, 1232, 1210, 1148,
1092, 1056, 1027 cmꢀ1 1H NMR (400 MHz, CDCl3): d 2.52 (d, 1H,
;
J = 6.6 Hz, OH), 3.53–3.60 (m, 2H, 2 ꢂ H-30), 3.67 (dd, 1H,
J = 5.0 Hz, J = 3.5 Hz, H-10), 3.97–4.03 (m, 1H, H-20), 4.09–4.12 (m,
1H, H-4), 4.45 (dd, 1H, J = 6.7 Hz, J = 5.1 Hz, H-5), 4.50 (m, 2H,
OCH2Ph), 4.67 (d, 1H, J = 11.5 Hz, OCH2Ph), 4.75 (d, 1H,
J = 11.5 Hz, OCH2Ph), 5.14 (d, 1H, J = 17.0 Hz, H-200trans), 5.19 (d,
1H, J = 10.1 Hz, H-200cis), 5.52 (s, 1H, NH), 5.74 (ddd, 1H,
J = 17.3 Hz, J = 10.1 Hz, J = 7.5 Hz, H-100), 7.29–7.38 (m, 10H, Ph);
13C NMR (100 MHz, CDCl3): d 57.0 (C-4), 69.1 (C-20), 70.9 (C-30),
73.5 (OCH2Ph), 74.4 (OCH2Ph), 77.3 (C-10), 81.5 (C-5), 118.9 (C-
200), 127.9 (3 ꢂ CHPh), 128.1 (CHPh), 128.4 (2 ꢂ CHPh), 128.5
(4 ꢂ CHPh), 135.7 (C-100), 137.4 (Ci), 137.7 (Ci), 158.3 (C-2). Anal.
Calcd for C22H25NO5: C, 68.91; H, 6.57; N, 3.65. Found: C, 68.99;
H, 6.61; N, 3.70.
159 °C; [a]
23 = +6.3 (c 0.24, CH3OH). IR (neat) mmax 3418, 3340,
D
3267, 3111, 2966, 2937, 2875, 2850, 2534, 2486, 2441, 1694,
1434, 1367, 1275, 1241, 1077, 1037 cmꢀ1; 1H NMR (400 MHz, CD3-
OD): d 0.99 (t, 3H, J = 7.4 Hz, CH3), 1.61–1.75 (m, 2H, CH2), 3.58 (dd,
1H, J = 9.7 Hz, J = 5.0 Hz, H-30), 3.62–3.71 (m, 2H, H-20, H-30), 3.81
(td, 1H, J = 8.2 Hz, J = 8.2 Hz, J = 5.1 Hz, H-4), 3.88–3.90 (m, 1H, H-
10), 4.74 (dd, 1H, J = 7.9 Hz, J = 4.7 Hz, H-5); 13C NMR (100 MHz,
CD3OD): d 11.1 (CH3), 24.2 (CH2), 58.1 (C-4), 64.0 (C-30), 69.9 (C-
10), 73.5 (C-20), 80.8 (C-5), 162.0 (C-2). Anal. Calcd for C8H15NO5:
C, 46.82; H, 7.37; N, 6.83. Found: C, 46.92; H, 7.48; N, 6.88.
4.1.11. N-[(30S,40S,50R,60R)-50,70-Bis(benzyloxy)-40,60-dihydroxy-
hept-10-en-30-yl]-2,2,2-trichloroacetamide 30
4.1.14. (4S,5R)-4-Ethyl-5-[(10Z)-pentadec-10-en-10-yl]oxazolidin-
2-one (Z)-32 and (4S,5R)-4-ethyl-5-[(10E)-pentadec-10-en-10-yl]
oxazolidin-2-one (E)-32
To a solution of 11 (0.20 g, 0.97 mmol) in MeOH (2.6 mL) was
added NaIO4 (0.52 g, 2.43 mmol) dissolved in water (2.6 mL) and
the resulting mixture was stirred at room temperature. After
30 min, the insoluble material was removed by filtration, the
filtrate was concentrated, and the residue was extracted with
EtOAc (3 ꢂ 10 mL). The combined organic extracts were dried over
Na2SO4, the solvent was evaporated, and the crude product was
used in the next reaction without further purification.
p-Toluenesulfonic acid (27 mg, 0.14 mmol) was added to a
solution of amide 16 (0.75 g, 1.38 mmol) in MeOH (27 mL) and
the resulting mixture was stirred at room temperature for 18 h,
before neutralization with Et3N. Removal of the solvent and chro-
matography of the residue through a short column of silica gel (n-
hexane/ethyl acetate, 2:1) furnished 0.68 g (98%) of diol 30 as a
colourless oil; [
a
]
27 = ꢀ12.8 (c 0.32, CHCl3). IR (neat) mmax 3356,
D
3030, 2867, 1701, 1644, 1497, 1454, 1407, 1361, 1215, 1072,
1026 cmꢀ1 1H NMR (400 MHz, CDCl3): d 2.72 (d, 1H, J = 3.8 Hz,
;
OH), 2.91 (d, 1H, J = 5.9 Hz, OH), 3.59 (dd, 1H, J = 9.7 Hz,
J = 5.0 Hz, H-70), 3.64 (dd, 1H, J = 9.7 Hz, J = 6.3 Hz, H-70),
3.70–3.72 (m, 1H, H-50), 3.93–3.96 (m, 1H, H-40), 4.05–4.11 (m,
1H, H-60), 4.50–4.62 (m, 4H, H-30, OCH2Ph), 4.72 (d, 1H,
J = 10.9 Hz, OCH2Ph), 5.28–5.34 (m, 2H, 2 ꢂ H-10), 5.81–5.89 (m,
1H, H-20), 7.27–7.38 (m, 10H, Ph), 7.59 (br d, 1H, J = 8.2 Hz, NH);
13C NMR (100 MHz, CDCl3): d 56.6 (C-30), 70.3 (C-60), 70.7 (C-70),
71.1 (C-40), 73.5 (OCH2Ph), 74.3 (OCH2Ph), 78.8 (C-50), 92.5 (C-2),
118.1 (C-10), 127.9 (2 ꢂ CHPh), 128.0 (CHPh), 128.3 (CHPh), 128.4
(2 ꢂ CHPh), 128.5 (2 ꢂ CHPh), 128.6 (2 ꢂ CHPh), 132.4 (C-20), 137.1
(Ci), 137.4 (Ci), 161.7 (C-1). Anal. Calcd for C23H26Cl3NO5: C,
54.94; H, 5.21; N, 2.79. Found: C, 55.31; H, 5.34; N, 2.85.
To a solution of 1,1,1,3,3,3-hexamethyldisilazane (0.58 mL,
2.73 mmol) in dry THF (2.9 mL) was added n-BuLi (1.70 mL,
2.72 mmol, a 1.6 M solution in n-hexane) at room temperature.
The solution of hexamethyldisilazide (LHMDS) obtained was trea-
ted with the known salt 1325 (1.67 g, 3.10 mmol), and the resulting
dark mixture was stirred for 5 min at the same temperature. Next,
a solution of the obtained aldehyde (0.14 g, 0.97 mmol) in dry THF
(2.9 mL) was added. After stirring for 30 min, the mixture was
diluted with EtOAc (25 mL) and poured into a saturated NH4Cl
solution (25 mL). The aqueous phase was washed with further por-
tions of EtOAc (2 ꢂ 17 mL). The combined organic layers were
dried over Na2SO4, and the solvent was evaporated, and the residue
was chromatographed on silica gel (n-hexane/ethyl acetate, 3:1) to
give 0.25 g (78%) of a barely separable mixture of olefins 32 as a
colourless oil. Repeated chromatography on silica gel (n-hexane/
ethyl acetate, 3:1) afforded only (Z)-32 in its pure form as an ana-
4.1.12. N-[(30R,40S,50R,60R)-50,70-Bis(benzyloxy)-40,60-dihydroxy-
hept-10-en-30-yl]-2,2,2-trichloroacetamide 31
Similar to the previous experiment, trichloroacetamide 17
(0.57 g, 1.05 mmol) and p-TsOH (21 mg, 0.11 mmol) provided after
stirring (20 h) and flash chromatography on silica gel (n-hexane/
ethyl acetate, 3:1) 0.51 g (96%) of compound 31 as a colourless
lytical sample. Alkene (Z)-32: [
a
]
24 = ꢀ4.2 (c 0.48, CHCl3). IR (neat)
D
mmax 3726, 3705, 3625, 3600, 3262, 2915, 2849, 1749, 1720, 1470,
1428, 1376, 1318, 1268, 1225, 1114, 1042, 996 cmꢀ1
;
1H NMR
oil; [a]
28 = +34.7 (c 0.60, CHCl3). IR (neat) mmax 3405, 3063, 3030,
D
(400 MHz, CDCl3): d 0.88 (t, 3H, J = 6.8 Hz, CH3), 0.93 (t, 3H,
Please cite this article in press as: Stanková, K.; et al. Tetrahedron: Asymmetry (2015), http://dx.doi.org/10.1016/j.tetasy.2015.11.001

12
K. Stanková et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
J = 7.4 Hz, CH3), 1.26–1.41 (m, 22H, 11 ꢂ CH2), 1.43–1.58 (m, 2H,
CH2), 1.98–2.17 (m, 2H, 2 ꢂ H-30), 3.72 (td, 1H, J = 8.2 Hz,
J = 8.2 Hz, J = 5.1 Hz, H-4), 5.38–5.42 (m, 1H, H-5), 5.51–5.56 (m,
1H, H-10), 5.76 (td, 1H, J = 10.6 Hz, J = 7.6 Hz, J = 7.6 Hz, H-20), 6.20
(s, 1H, NH); 13C NMR (100 MHz, CDCl3): d 10.3 (CH3), 14.3 (CH3),
22.7 (CH2), 24.0 (CH2), 27.8 (C-30), 29.2 (CH2), 29.3 (CH2), 29.4
(CH2), 29.5 (CH2), 29.6 (2 ꢂ CH2), 29.7 (3 ꢂ CH2), 31.9 (CH2), 57.6
(C-4), 75.9 (C-5), 122.5 (C-10), 137.0 (C-20), 159.7 (C-2). Anal. Calcd
for C20H37NO2: C, 74.25; H, 11.53; N, 4.33. Found: C, 74.31; H,
11.59; N, 4.37.
34 as a colourless oil; [
1026, 1060, 1088, 1180, 1202, 1227, 1319, 1360, 1411, 1454,
1496, 1747, 2866, 3029, 3062 cmꢀ1 1H NMR (400 MHz, CDCl3): d
a]
24 = +77.0 (c 0.40, CHCl3). IR (neat) mmax
D
;
3.45 (dd, 1H, J = 6.1 Hz, J = 2.3 Hz, H-10), 3.65–3.73 (m, 2H, H-4,
H-30), 3.80 (dd, 1H, J = 11.0 Hz, J = 2.5 Hz, H-30), 3.87 (d, 1H,
J = 15.1 Hz, NCH2Ph), 3.94 (td, 1H, J = 5.8 Hz, J = 5.8 Hz, J = 2.5 Hz,
H-20), 4.25 (dd, 1H, J = 7.3 Hz, J = 2.3 Hz, H-5), 4.41 (d, 1H,
J = 11.8 Hz, OCH2Ph), 4.48 (d, 1H, J = 12.0 Hz, OCH2Ph), 4.55 (d,
1H, J = 12.1 Hz, OCH2Ph), 4.56 (d, 1H, J = 11.8 Hz, OCH2Ph), 4.65–
4.71 (m, 3H, H-200trans, OCH2Ph), 4.75 (d, 1H, J = 15.1 Hz, NCH2Ph),
5.15 (d, 1H, J = 10.0 Hz, H-200cis), 5.51 (ddd, 1H, J = 17.0 Hz,
J = 9.9 Hz, J = 9.2 Hz, H-100), 6.94–6.95 (m, 2H, Ph), 7.17–7.37 (m,
18H, Ph); 13C NMR (100 MHz, CDCl3): d 45.7 (NCH2Ph), 59.8 (C-
4), 70.0 (C-30), 73.0 (OCH2Ph), 73.4 (2 ꢂ OCH2Ph), 75.3 (C-10), 77.6
(C-5), 78.5 (C-20), 121.4 (C-200), 127.6 (2 ꢂ CHPh), 127.7 (3 ꢂ CHPh),
127.8 (CHPh), 127.9 (4 ꢂ CHPh), 128.3 (2 ꢂ CHPh), 128.4 (4 ꢂ CHPh),
128.5 (2 ꢂ CHPh), 128.6 (2 ꢂ CHPh), 134.6 (C-100), 135.7 (Ci), 137.5
(Ci), 138.1 (Ci), 138.3 (Ci), 157.5 (C-2). Anal. Calcd for C36H37NO5:
C, 76.71; H, 6.62; N, 2.48. Found: C, 76.80; H, 6.71; N, 2.54.
4.1.15. (4S,5R)-4-Ethyl-5-pentadecyloxazolidin-2-one 33
A solution of 32 (0.10 g, 0.309 mmol) in dry EtOAc (6.2 mL) was
treated with 5% Rh in Al2O3 (15.6 mg) and the resulting suspension
was stirred at room temperature under an atmosphere of hydro-
gen. After 3 h, the mixture was filtered through a small pad of
Celite, the filtrate was concentrated in vacuo, and the residue
was chromatographed on silica gel (n-hexane/ethyl acetate, 3:1)
to give 75 mg (75%) of compound 33 as a white amorphous solid;
[a]
24 = +5.1 (c 0.76, CHCl3). IR (neat) mmax 3264, 3145, 2914, 2848,
D
1754, 1466, 1429, 1391, 1229, 1100, 1039 cmꢀ1
;
1H NMR
4.1.18. (4R,5S)-3-Benzyl-4-ethyl-5-[(10S,20R)-10,20,30-trihydroxy-
propyl]oxazolidin-2-one 12
(400 MHz, CDCl3): d 0.88 (t, 3H, J = 6.8 Hz, CH3), 0.96 (t, 3H,
J = 7.4 Hz, CH3), 1.11–1.34 (m, 26H, 13 ꢂ CH2), 1.43–1.47 (m, 4H,
2 ꢂ CH2), 3.65 (ddd, 1H, J = 9.4 Hz, J = 7.6 Hz, J = 4.3 Hz, H-4),
4.50–4.60 (m, 1H, H-5), 5.75 (s, 1H, NH); 13C NMR (100 MHz,
CDCl3): d 10.5 (CH3), 14.1 (CH3), 22.7 (CH2), 22.9 (CH2), 25.9
(CH2), 29.1 (CH2), 29.4 (3 ꢂ CH2), 29.5 (CH2), 29.6 (3 ꢂ CH2), 29.7
(3 ꢂ CH2), 31.9 (CH2), 57.2 (C-4), 80.4 (C-5), 159.5 (C-2). Anal. Calcd
for C20H39NO2: C, 73.79; H, 12.08; N, 4.30. Found: C, 73.85; H,
12.14; N, 4.35.
At first, 10% Pd/C (80 mg) was added to a solution of 34 (0.50 g,
0.887 mmol) in EtOH (7 mL) and the reaction mixture was stirred
at room temperature under an atmosphere of hydrogen. After
2 h, the catalyst was removed by filtration through a small pad of
Celite, the solvent was evaporated, and the residue was
chromatographed on silica gel (ethyl acetate/MeOH, 100:1) to give
0.22 g (83%) of derivative 12 as a colourless oil; [
(c 0.26, CHCl3). IR (neat) mmax 1029, 1054, 1099, 1203, 1231,
1261, 1361, 1389, 1441, 1496, 1716, 2930, 3367 cmꢀ1 1H NMR
a]
23 = +74.2
D
;
4.1.16. (3S,4R)-3-Aminononadecan-4-ol 712c
(400 MHz, CDCl3): d 0.80 (t, 3H, J = 7.4 Hz, CH3), 1.45–1.64 (m,
2H, CH2), 4.53 (dd, 1H, J = 4.4 Hz, J = 2.9 Hz, H-10), 3.62–3.70 (m,
2H, H-30, H-4), 3.74 (dd, 1H, J = 11.7 Hz, J = 3.9 Hz, H-30), 3.83–
3.87 (m, 1H, H-20), 4.12 (d, 1H, J = 15.5 Hz, NCH2Ph), 4.31 (dd, 1H,
J = 5.3 Hz, J = 2.9 Hz, H-5), 4.68 (d, 1H, J = 15.5 Hz, NCH2Ph), 7.22–
7.32 (m, 5H, Ph); 13C NMR (100 MHz, CDCl3): d 7.6 (CH3), 24.1
(CH2), 46.1 (NCH2Ph), 57.2 (C-4), 63.7 (C-30), 71.7 (C-20), 72.4 (C-
10), 78.6 (C-5), 127.8 (3 ꢂ CHPh), 128.7 (2 ꢂ CHPh), 135.5 (Ci),
158.4 (C-2). Anal. Calcd for C15H21NO5: C, 61.00; H, 7.17; N, 4.74.
Found: C, 61.15; H, 7.29; N, 4.81.
A solution of 33 (75 mg, 0.23 mmol) in dry EtOH (4.6 mL) was
treated with a 2 M aq NaOH solution (4.6 mL) and the resulting
mixture was stirred and heated at reflux. After 5 h, no starting
material was identified in the mixture (as judged by TLC), which
was then concentrated in vacuo. The residue obtained was
extracted with CH2Cl2 (3 ꢂ 15 mL). The combined organic layers
were dried over Na2SO4, the solvent was evaporated, and the crude
product was chromatographed on silica gel (CH2Cl2/MeOH, 2:1) to
furnish 63 mg (91%) of compound 712c as a white solid; mp
74–75 °C (recrystallized from ethyl acetate/MeOH); lit.12c mp not
reported;
MeOH); lit.12c
1573, 1469, 1076 cmꢀ1
[
a
]
24 = +18.8 (c 0.33, CHCl3);
[a]
24 = +14.0 (c 0.35,
4.1.19. (4R,5R)-3-Benzyl-4-ethyl-5-[(10Z)-pentadec-10-en-10-yl]
oxazolidin-2-one (Z)-35 and (4R,5R)-3-Benzyl-4-ethyl-5-[(10E)-
pentadec-10-en-10-yl]oxazolidin-2-one (E)-35
D
D
[a] not reported. IR (neat) mmax 2956, 2914, 2848,
D
;
1H NMR (400 MHz, CD3OD): d 0.89 (t,
3H, J = 6.8, CH3), 0.97 (t, 3H, J = 7.5, CH3), 1.28–1.62 (m, 30H,
15 ꢂ CH2), 2.60 (dt, 1H, J = 8.6 Hz, J = 4.4 Hz, J = 4.2 Hz, H-3),
3.45–3.49 (m, 1H, H-4); 13C NMR (100 MHz, CD3OD): d 11.2
(CH3), 14.5 (CH3), 23.8 (CH2), 25.4 (CH2), 27.3 (CH2), 30.5 (CH2),
30.8 (9 ꢂ CH2), 33.0 (CH2), 33.1 (CH2), 58.7 (C-3), 75.2 (C-4). Anal.
Calcd for C19H41NO: C, 76.19; H, 13.80; N, 4.68. Found: C, 76.29;
H, 13.86; N, 4.74.
To a solution of 12 (0.2 g, 0.677 mmol) in MeOH (1.3 mL) was
added NaIO4 (0.365 g, 1.7 mmol) dissolved in water (1.3 mL) and
the resulting mixture was stirred at room temperature. After
30 min, the mixture was diluted with CH2Cl2 (4 mL), the insoluble
parts were filtered off, and the filtrate was concentrated in vacuo.
The obtained residue was washed with CH2Cl2 (3 ꢂ 15 mL), the
combined organic extracts were dried over Na2SO4, the solvent
was evaporated, and the crude product was used in the next reac-
tion without further purification.
To a solution of 1,1,1,3,3,3-hexamethyldisilazane (0.425 mL,
1.9 mmol) in dry THF (2.2 mL) was added n-BuLi (1.19 mL,
1.9 mmol, a 1.6 M solution in n-hexane) at room temperature.
The solution of hexamethyldisilazide (LHMDS) obtained was trea-
ted with the known salt 1325 (1.17 g, 2.17 mmol), and the resulting
dark mixture was stirred for 5 min at the same temperature. Next,
a solution of the obtained aldehyde (0.157 g, 0.673 mmol) in dry
THF (2.2 mL) was added. After stirring for 30 min, the mixture
was diluted with EtOAc (20 mL) and poured into a saturated
NH4Cl solution (14 mL). The aqueous phase was washed with
further portions of EtOAc (2 ꢂ 20 mL). The combined organic
extracts were dried over Na2SO4, the solvent was evaporated, and
4.1.17. (4R,5S)-3-Benzyl-5-[(10R,20R)-10,20,30-tris(benzyloxy)pro-
pyl]-4-vinyloxazolidin-2-one 34
To a solution of 29 (0.40 g, 1.04 mmol) in dry DMF (2.5 mL),
which had been cooled to 0 °C, were successively added NaH
(0.10 g, 4.17 mmol, 60% dispersion in mineral oil), BnBr (0.30 mL,
2.5 mmol) and TBAI (7.4 mg, 0.02 mmol). The resulting mixture
was stirred for 10 min at 0 °C and then at room temperature for
another 20 min. The excess hydride was decomposed by the cau-
tious addition of MeOH, after which the mixture was poured into
ice-water (25 mL) and extracted with Et2O (2 ꢂ 25 mL). The
combined organic layers were dried over Na2SO4, the solvent was
taken down, and the residue was chromatographed on silica gel
(n-hexane/ethyl acetate, 4:1) to afford 0.56 g (95%) of compound
Please cite this article in press as: Stanková, K.; et al. Tetrahedron: Asymmetry (2015), http://dx.doi.org/10.1016/j.tetasy.2015.11.001

K. Stanková et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
13
the residue was chromatographed on silica gel (n-hexane/ethyl
acetate, 9:1) to give 0.23 g (82%) of a barely separable mixture of
olefins 35 as a colourless oil. Repeated chromatography on silica
gel (n-hexane/ethyl acetate, 9:1) afforded only (Z)-35 in its pure
form as an analytical sample. Alkene (Z)-35: colourless oil;
the residue on silica gel (CH2Cl2/MeOH, 20:1) gave 8.41 g (79%)
of compound 1325 as a white solid; mp 75–76 °C (recrystallized
from Et2O); lit.25a mp not reported; lit.25b mp 83–84 °C. IR (neat)
mmax 2918, 2850, 1586, 1483, 1467, 1436, 1316, 1184, 1160,
1112, 1027 cmꢀ1
;
1H NMR (400 MHz, CDCl3): d 0.87 (t, 3H,
[
a]
D
24 = +30.4 (c 0.50, CHCl3). IR (neat) mmax 1003, 1048, 1102,
J = 6.8 Hz, CH3), 1.19–1.31 (m, 20H, 10 ꢂ CH2), 1.62–1.63 (m, 4H,
2 ꢂ CH2), 3.72–3.79 (m, 2H, CH2), 7.69–7.74 (m, 6H, Ph), 7.79–
7.87 (m, 9H, Ph); 13C NMR (100 MHz, CDCl3): d 14.0 (CH3), 22.5
(CH2), 22.6 (CH2), 22.9 (CH2), 29.1 (2 ꢂ CH2), 29.2 (CH2), 29.4
(CH2), 29.5 (CH2), 29.6 (2 ꢂ CH2), 30.3 (CH2), 30.4 (CH2), 31.8
(CH2), 117.9 (2 ꢂ Ci), 118.7 (Ci), 130.3 (3 ꢂ CHPh), 130.5
(3 ꢂ CHPh), 133.5 (3 ꢂ CHPh), 133.6 (3 ꢂ CHPh), 134.9 (3 ꢂ CHPh).
Anal. Calcd for C32H44BrP: C, 71.23; H, 8.22. Found: 71.30; H, 8.16.
1179, 1200, 1224, 1258, 1308, 1359, 1414, 1456, 1496, 1750,
2851, 2921 cmꢀ1 1H NMR (400 MHz, CDCl3): d 0.84–0.90 (m, 6H,
;
2 ꢂ CH3), 1.22–1.42 (m, 22H, 11 ꢂ CH2), 1.51–1.71 (m, 2H, CH2),
2.02–2.20 (m, 2H, 2 ꢂ H-30), 3.21 (td, 1H, J = 7.0 Hz, J = 7.0 Hz,
J = 3.2 Hz, H-4), 4.05 (d, 1H, J = 15.3 Hz, NCH2Ph), 4.81 (d, 1H,
J = 15.3 Hz, NCH2Ph), 4.85–4.89 (m, 1H, H-5), 5.32–5.38 (m, 1H,
H-10), 5.67 (ddd, 1H, J = 10.8 Hz, J = 7.6 Hz, J = 7.6 Hz, H-20), 7.26–
7.37 (m, 5H, Ph); 13C NMR (100 MHz, CDCl3): d 7.9 (CH3), 14.1
(CH3), 22.7 (CH2), 23.4 (CH2), 27.8 (C-30), 29.2 (CH2), 29.3
(2 ꢂ CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (2 ꢂ CH2), 29.7 (2 ꢂ CH2),
31.9 (CH2), 46.0 (NCH2Ph), 61.1 (C-4), 73.8 (C-5), 126.0 (C-10),
127.8 (CHPh), 128.0 (2 ꢂ CHPh), 128.7 (2 ꢂ CHPh), 136.0 (Ci), 137.1
(C-20), 158.2 (C-2) Anal. Calcd for C27H43NO2: C, 78.40; H, 10.48;
N, 3.39. Found: C, 78.51; H, 10.55; N, 3.43.
4.1.23. X-ray techniques
A single crystal of 15 suitable for X-ray diffraction was obtained
from n-hexane by slow evaporation at room temperature. The
intensities were collected at 173 K on an Oxford Diffraction XCal-
ibur2 CCD diffractometer using Mo K
a radiation (k = 0.71073 Ǻ).
Selected crystallographic and other relevant data for compound
15 are listed in Table 3. The structure was solved by direct meth-
ods.35 All non-hydrogen atoms were refined anisotropically by
full-matrix least squares calculations based on F2.35 All hydrogen
atoms were included in calculated positions as riding atoms,
with SHELXL9735 defaults. The PLATON36 programme was used
for structure analysis and molecular and crystal structure
drawings.
4.1.20. (4R,5R)-4-Ethyl-5-pentadecyloxazolidin-2-one 36
To a solution of olefins 35 (80 mg, 0.19 mmol) in dry EtOH
(2.9 mL) was added 10% Pd/C (20 mg) and the resulting suspension
was stirred at room temperature under an atmosphere of hydro-
gen. After 2 h, the mixture was diluted with EtOH (12.9 mL) and
36% HCl (0.2 mL) was added followed by the addition of another
portion of 10% Pd/C (37 mg). The resulting mixture was stirred
and heated at 60 °C for 8 h. The catalyst was filtered through a
small pad of Celite, after which the filtrate was concentrated in
vacuo, and the residue was chromatographed on silica gel (n-hex-
ane/ethyl acetate, 3:1) to afford 31 mg (50%) of compound 36 as a
4.1.24. Supplementary data
Complete crystallographic data for the structural analysis have
been deposited with the Cambridge Crystallographic Data Centre,
CCDC No. 1032389 for compound 15. These data can be obtained
free of charge from the Director, Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44 1223
336033; e-mail: deposit@ccdc.cam.ac.uk or via: www.ccdc.cam.
ac.uk).
white amorphous solid; [a]
24 = +40.0 (c 0.16, CHCl3). IR (neat) mmax
D
3727, 3704, 3263, 2956, 2915, 2848, 1774, 1736, 1464, 1397, 1293,
1258, 1244, 1157, 1092, 1040, 1010, 977 cmꢀ1; 1H NMR (400 MHz,
CDCl3): d 0.88 (t, 3H, J = 6.8 Hz, CH3), 0.95 (t, 3H, J = 7.5 Hz, CH3),
1.26–1.77 (m, 30H, 15 ꢂ CH2), 3.35–3.39 (m, 1H, H-4), 4.16 (dt,
1H, J = 7.8 Hz, J = 5.2 Hz, J = 5.2 Hz, H-5), 6.17 (s, 1H, NH); 13C
NMR (100 MHz, CDCl3): d 9.5 (CH3), 14.1 (CH3), 22.7 (CH2), 24.7
(CH2), 28.2 (CH2), 29.3 (2 ꢂ CH2), 29.4 (CH2), 29.5 (CH2), 29.6
(3 ꢂ CH2), 29.7 (3 ꢂ CH2), 31.9 (CH2), 34.9 (CH2), 59.2 (C-4), 82.3
(C-5), 159.5 (C-2). Anal. Calcd for C20H39NO2: C, 73.79; H, 12.08;
N, 4.30. Found: C, 73.85; H, 12.14; N, 4.35.
4.2. Antiproliferative/cytotoxic activity
4.2.1. Cell culture
The following human cancer cell lines were used for this study:
MCF-7 and MDA-MB-231 (breast cancer cells), Jurkat (human
acute T-lymphoblastic leukaemia), HCT-116 (human colon carci-
noma) and Caco-2 (human colon carcinoma) and non-cancerous
cell line NiH 3T3 (mouse fibroblasts). MCF-7, HCT-116, Caco-2
and Jurkat cells were maintained in RPMI 1640 medium. MDA-
MB-231 and NiH 3T3 cell lines were maintained in growth medium
consisting of high glucose Dulbecco’s Modified Eagle’s Medium.
Both of these media were supplemented with Glutamax, and with
10% (V/V) foetal calf serum, penicillin (100 IU mLꢀ1), and strepto-
mycin (100 mg mLꢀ1) (all from Invitrogen, Carlsbad, CA USA), in
the atmosphere of 5% CO2 in humidified air at 37 °C. Cell viability,
estimated by the Trypan blue exclusion, was greater than 95%
before each experiment.
4.1.21. (3R,4R)-3-Aminononadecan-4-ol 10
Using the same procedure as for the preparation of 7, com-
pound 36 (50 mg, 0.15 mmol) provided, after treatment (6 h) with
a 2 M aq NaOH solution (3 mL) in EtOH (3 mL) and chromatogra-
phy on silica gel (CH2Cl2/MeOH, 2:1), 43 mg (92%) of derivative
10 as a white solid; mp 45–47 °C (recrystallized from ethyl acet-
ate/MeOH); [
3291, 2955, 2916, 2848, 1607, 1463, 1347, 1278, 1147, 1099,
1034, 946 cmꢀ1 1H NMR (400 MHz, CD3OD): d 0.90 (t, 3H,
a]
23 = +12.7 (c 0.22, CHCl3). IR (neat) mmax 3364,
D
;
J = 6.8 Hz, CH3), 0.96 (t, 3H, J = 7.5 Hz, CH3), 1.12–1.63 (m, 30H,
15 ꢂ CH2), 2.48 (dt, 1H, J = 7.9 Hz, J = 5.1 Hz, J = 5.1 Hz, H-3),
3.34–3.37 (m, 1H, H-4); 13C NMR (100 MHz, CD3OD): d 11.0
(CH3), 14.5 (CH3), 23.8 (CH2), 27.1 (2 ꢂ CH2), 30.5 (CH2), 30.8
(9 ꢂ CH2), 33.1 (CH2), 34.8 (CH2), 58.2 (C-3), 74.7 (C-4). Anal. Calcd
for C19H41NO: C, 76.19; H, 13.80; N, 4.68. Found: C, 76.29; H, 13.86;
N, 4.74.
4.2.2. Cytotoxicity assay
The cytotoxic effect of the tested compounds was studied using
the colorimetric microculture assay with the MTT endpoint.37 The
amount of MTT reduced to formazan was proportional to the num-
ber of viable cells. Briefly, 5 ꢂ 103 cells were plated per well in 96-
well polystyrene microplates (Sarstedt, Germany) in the culture
medium containing tested chemicals at final concentrations 10ꢀ4
4.1.22. Triphenyl(tetradecyl)phosphonium bromide 1325
To
a
solution of commercial 1-bromotetradecane (5.47 g,
to 10ꢀ6 mol Lꢀ1. After 72 h of incubation, 10
were added into each well. After an additional 4 h, during which
insoluble formazan was formed, 100 L of 10% (m/m) sodium
dodecyl sulfate was added into each well and another 12 h were
l )
L of MTT (5 mg mLꢀ1
19.73 mmol) in dry toluene (16.5 mL) was added Ph3P (6.21 g,
23.67 mmol), and the resulting mixture was stirred and heated at
reflux for 24 h. Removal of the solvent and chromatography of
l
Please cite this article in press as: Stanková, K.; et al. Tetrahedron: Asymmetry (2015), http://dx.doi.org/10.1016/j.tetasy.2015.11.001

14
K. Stanková et al. / Tetrahedron: Asymmetry xxx (2015) xxx–xxx
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Acknowledgements
The present work was supported by the Grant Agency (No.
1/0168/15, No. 1/0398/14 and No. 1/0322/14) of the Ministry of
Education, Slovak Republic. It was also supported by the Slovak
Research and Development Agency (No. APVV-14-0883), VVES-
2014-172 and by the project MediPark Košice: 26220220185 sup-
ported by Operational Programme Research and Development (OP
VaV-2012/2.2/08-RO, contract No. OPVaV/12/2013). We thank
Dr. Juraj Kuchár (P. J. Šafárik University, Košice) for his assistance
in X-ray measurements.
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