The convergent synthesis and anticancer activity of broussonetinines related analogues

Article abstract of DOI:10.1016/j.carres.2017.09.009

The convergent synthesis of broussonetinines related congeners 3 and 4 with the simple C₁₃ alkyl side chain and differently configured pyrrolidine skeleton has been achieved. Our approach relied on the [3,3]-sigmatropic rearrangements of chiral allylic substrates derived from D-xylose. Cross metathesis of the common oxazolidinone intermediates 7 and 8 with tridec-1-ene followed by alkylative cyclization completed the construction of both C-alkyl iminosugars. The targeted compounds 3 and 4 were screened for antiproliferative/cytotoxic activities against multiple cancer cell lines by MTT assay. Compound 3 exhibited very good in vitro potency on Caco-2 and Jurkat cell lines with IC₅₀ value of 5.1 μM and 5.8 μM, respectively.

Full text of DOI:10.1016/j.carres.2017.09.009

Carbohydrate Research 451 (2017) 59e71
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
The convergent synthesis and anticancer activity of broussonetinines
related analogues
,
*
a
ꢀ a
ꢀ
ꢀ a
ꢀ a
Dominika Jackova , Miroslava Martinkova , Jozef Gonda , Kvetoslava Stankova ,
c
,
d
c
ꢀ b
ꢁ ꢁ
Martina Bago Pilatova , Peter Herich , Jozef Kozísek
Institute of Chemical Sciences, Department of Organic Chemistry, Faculty of Science, P.J. Safarik University, Moyzesova 11, 040 01 Kosice, Slovak Republic
Institute of Pharmacology, Faculty of Medicine, P.J. Safarik University, SNP 1, 040 66 Kosice, Slovak Republic
Institute of Physical Chemistry and Chemical Physics, Department of Physical Chemistry, Slovak University of Technology, Radlinskeho 9, 812 37 Bratislava,
a
ꢁ
ꢀ
ꢁ
b
c
ꢁ
ꢀ
ꢁ
ꢀ
Slovak Republic
d
ꢀ
Central Laboratories, Slovak University of Technology, Radlinskeho 9, 812 37 Bratislava, Slovak Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
The convergent synthesis of broussonetinines related congeners 3 and 4 with the simple C13 alkyl side
chain and differently configured pyrrolidine skeleton has been achieved. Our approach relied on the
[3,3]-sigmatropic rearrangements of chiral allylic substrates derived from D-xylose. Cross metathesis of
the common oxazolidinone intermediates 7 and 8 with tridec-1-ene followed by alkylative cyclization
completed the construction of both C-alkyl iminosugars. The targeted compounds 3 and 4 were screened
for antiproliferative/cytotoxic activities against multiple cancer cell lines by MTT assay. Compound 3
Received 5 September 2017
Received in revised form
14 September 2017
Accepted 14 September 2017
Available online 19 September 2017
exhibited very good in vitro potency on Caco-2 and Jurkat cell lines with IC50 value of 5.1
respectively.
mM and 5.8 mM,
Keywords:
Broussonetines
Broussonetinines
© 2017 Elsevier Ltd. All rights reserved.
[3,3]-Sigmatropic rearrangements
Oxazolidinones pyrrolidine unit
Metathesis
Antiproliferative/cytotoxic activity
1. Introduction
In our continuing studies based on the feasibility of the [3,3]-
sigmatropic rearrangements in the total synthesis of sphingoid
base-like natural products and their analogues [10] we were
interested in investigating the use of such transformation as the key
reaction for the construction of broussonetinines related conge-
ners, which possess the simple C13 hydrocarbon fragment. Herein,
we would like to report the total synthesis and antiproliferative
Broussonetines, along with diastereomeric broussonetinines,
the representative structures of which are illustrated by broussso-
netine C (1) and broussonetinine A (2), were isolated by Kusano and
co-workers [1] from the branches of the Asian deciduous tree
Broussonetia kazinoki (Fig.1). They represent a class of more than 30
well-identified and characterized polyhydroxylated pyrrolidine
alkaloids, which possess variable side chains with the diverse types
of functionalization [1]. Most of these C-alkyl iminosugars
demonstrated significant glycosidase inhibitory activities with IC50
values in the micromolar to nanomolar range [1e3] and as such are
expected to be promising anticancer [4], antidiabetic [5], antiviral
[6] and anti-inflammatory agents [7], and pharmacological chap-
erones [8]. Due to these potent biological properties, several syn-
thetic methods towards naturally occurring broussonetines [2,3,9]
and their related analogues [2-3,8b,9d] have been reported.
activity of two iminosugars 3 and 4, starting from D-xylose. While
this manuscript was in preparation, we published the approach
towards other diastereomeric analogues of our final compounds 3 a
4 together with their cytotoxic profile [11]. Part of the reported
synthetic strategy [11] is here effectively applied.
2. Results and discussion
2.1. Chemistry
As shown in Fig. 2, our retrosynthetic route to broussonetinines
analogues 3 and 4 would involve pyrrolidine core construction
through the intramolecular SN2 cyclization of open chain scaffolds
5 and 6, respectively. We planned to install the C13 side segment in
* Corresponding author.
E-mail address: miroslava.martinkova@upjs.sk (M. Martinkova).
ꢀ
https://doi.org/10.1016/j.carres.2017.09.009
0008-6215/© 2017 Elsevier Ltd. All rights reserved.

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60
D. Jackova et al. / Carbohydrate Research 451 (2017) 59e71
Fig. 1. Structures of broussonetine C (1) and broussonetinine A (2).
Fig. 2. Retrosynthetic route towards broussonetinines related analogues 3 and 4.
5 and 6 via cross metathesis chemistry of the common oxazolidi-
nones 7 and 8, which could be constructed by the [3,3]-sigmatropic
rearrangements of the allylic substrates 9 and 10 derived from D-
unambiguously supported an (E)-relationship between vinyl pro-
tons). It should be noted that no trace of the second geometric
isomer was detected in the crude 1H NMR spectrum. The lower
yield is most likely due to generation of the minor unidentified side
products. Further attempts to improve the efficiency of the olefi-
nation turned out to be ineffective and afforded 16 in the similar or
lower amounts. After protection of the diol moiety in 16 with 2,2-
DMP, the ester functionality in the created acetonide 17 (97%)
was then reduced with DIBAl-H to give the corresponding alcohol
18 [14] (96%, 43% overall yield starting from 11).
With the construction of the allylic scaffold 18 accomplished
(Scheme 1), we were now in a position to examine the key [3,3]-
sigmatropic rearrangements. For this purpose, compound 18 was
converted into the appropriate substrates 9 and 10, respectively.
Thiocyanate 9 (93%) was produced via the known [10d] two-step
approach involving the formation of a mesylate intermediate. On
the other hand, the corresponding imidate 10 was prepared by
treatment of 18 with Cl3CCN and catalytic amounts of DBU and was
used immediately in the Overman transformation [15] without
purification (Scheme 2). The microwave-promoted thermal aza-
xylose.
The commercially available
D-xylose was modified into the
known 1,2-O-isopropylidene-5-O-trityl-
a-D-xylofuranose 11 [12b]
according to the literature [12] (Scheme 1). The conversion of the
C-3 stereochemistry in 11 was achieved through an oxidation-
highly diastereoselective reduction protocol, affording the corre-
sponding 1,2-O-isopropylidene-5-O-trityl-
in 92% yield over two steps via formation of the 1,2-O-iso-
propylidene-5-O-trityl- -erythro-pentofuranos-3-ulose [13].
a-D-ribofuranose 12 [13]
a-D
Removal of the O-trityl protecting group in 12 (CSA, MeOH/CH2Cl2)
and subsequent treatment of the resulting diol 13 [14] (96%) with
BnBr produced 3,5-di-O-benzyl-1,2-O-isopropylidene-a-D-ribofur-
anose 14 [14] in 94% yield from 12. Exposure of 14e80% TFA
resulted in cleavage of the isopropylideneketal fragment to provide
lactol 15 [14] (80%) as a mixture of anomers. Wittig reaction of 15
with the stable ylide reagent (Ph3P ¼ CHCO2Et) produced (E)-
a,b-
unsaturated ester 16 (67%, J ¼ 15.7 Hz, this coupling constant value
Scheme 1. Reagents and conditions: (a) IBX, MeCN, reflux, 93%; (b) NaBH4, EtOH/CH2Cl2, 0 ꢀC/rt, 99%; (c) CSA, MeOH/CH2Cl2, rt; (d) BnBr, NaH, DMF, TBAI, 0 ꢀC/rt; (e) 80% TFA, rt;
(f) Ph3P ¼ CHCO2Et, CH2Cl2, benzoic acid, rt; (g) 2,2-DMP, p-TsOH, CH2Cl2, rt; (h) DIBAl-H, CH2Cl2, ꢁ30 ꢀC.

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D. Jackova et al. / Carbohydrate Research 451 (2017) 59e71
61
Scheme 2. Reagents and conditions: (a) (i) MsCl, Et3N, CH2Cl2, 0 ꢀC/rt; (ii) KSCN, MeCN, 0 ꢀC/rt; (b) Cl3CCN, DBU, CH2Cl2, 0 ꢀC/rt; (c) n-heptane, 90 ꢀC,
o-xylene, K2CO3, MW, 150 ꢀC (85%) or 170 ꢀC (94%).
D
(60%) or MW (77%); (d)
Claisen rearrangement of 9 was carried out in n-heptane at 90 ꢀC to
produce the rearranged products 19 and 20 in good combined yield
(77%) after 1 h, as a separable mixture of diastereoisomers
(19:20 ¼ 48:52, the ratio was determined by 1H NMR spectroscopic
analysis). The rearrangement reaction of 9 realized under the
conventional heating (6 h) at the same temperature allowed the
preparation of both isothiocyanates 19 and 20 with lower yield
(60%) and similar selectivity (19:20 ¼ 44:56). In both cases we also
recovered the starting thiocyanate 9 (11e13%). The thermal Over-
man rearrangement of 10 (Scheme 2) mediated by microwave
irradiation was conducted in o-xylene in the presence of K2CO3 [16]
either at 150 ꢀC (3.5 h) or 170 ꢀC (1 h) and provided an inseparable
mixture of the corresponding trichloroacetamides 21 and 22
(21:22 ¼ 44:56 or 56:44 in both cases) in very good combined
yields 85% and 94%, respectively, and with diastereoselectivities
similar to those observed in the [3,3]-sigmatropic rearrangement of
9. Although both used rearrangement reactions afforded an un-
satisfactory dr, the relative short-step approach to the aforemen-
tioned intermediates 19e22 from 11 with respectable overall yields
as well as and a possibility to demonstrate approach towards the
new broussonetinines analogues led us to continue in our elabo-
rated strategy.
7, the observed NOE interactions between H-4 and H-5 (1%)
unambiguously establish their trans orientation revealing the
opposite (4R)-configuration. Moreover, the configuration of the
above mentioned newly constructed stereocentre in 24 was
assigned to be (4R) through X-ray analysis (Fig. 3 and Table 1) of the
suitable crystal which was provided by its recrystallization from a
mixture of n-hexane and ethyl acetate, confirming that the rear-
ranged product 19 must have the same stereochemistry.
Having successfully resolved the stereochemical assignment, we
directed our efforts towards construction of the required pyrroli-
dine unit with the C-13 hydrophobic tail with the aim to obtain the
final analogues 3 and 4, and this was readily achieved by using the
strategy depicted in Scheme 4. Formation of the N-Boc derivatives
26 (78%) and 27 (89%) from the corresponding carbamates 7 and 8
proceeded without problem [17]. It should be noted that during
tert-butoxycarbonylation of 7 and 8 we also isolated the corre-
sponding di-Boc derivative in 10% and 9% yields, respectively. After
protection of the secondary hydroxy substituent in 26 and 27 as a
tosyloxy group (TsCl, DMAP, Et3N, Me3N.HCl) [17], the subsequent
hydrolysis of the oxazolidinone skeleton of 28 (95%) and 29 (87%)
produced the amino alcohols 30 and 31 in 81% and 92% yields,
respectively (Scheme 4). At this stage, the installation of the long
alkyl chain through Grubbs' cross metathesis was performed and
the aforementioned advanced vinyl scaffolds 30 and 31 were sub-
jected to the coupling reaction with tridec-1-ene to afford the (E)-
configured olefin 5 (79%, J ¼ 15.6 Hz) and 6 (79%) as the sole
product in both cases (Scheme 4). In the case of 6 it was not possible
to determine correctly the value of the coupling constant of the
vinyl protons. Therefore, the geometry of the double bond was
unambiguously confirmed by 1H NMR analysis of derivative 33
(vide infra). With the requisite alkenes 5 and 6 in hand, we then
focused our attention on the preparation of the crucial pyrrolidine
unit by adopting the recently reported Marco's protocol [9f]. Thus,
removal of the temporary Boc protecting group of 5 and 6 followed
by work up with aq NH3 delivered the expected cyclic products 32
(96%) and 33 (86%, J ¼ 15.2 Hz). Finally, the simultaneous saturation
of the double bond and deprotection of the O-benzyl groups in 32
and 33 were achieved via catalytic hydrogenation to furnish the
targeted broussonetinines congeners 3 and 4 in the nearly quan-
titative yields. The relative configuration of 3 was confirmed by 1H
NMR NOE analysis (Scheme 4). The observed larger enhancements
between H-2 and H-3, between H-3 and H-4 and between H-4 and
H-5 protons revealed that they all occupy the same face of the
pyrrolidine skeleton. In the case of compound 4, due to the overlap
of the H-4 proton signal with the signals of the hydroxymethyl side
The configuration of the newly generated stereocentre with
nitrogen was confirmed by NOESY experiments which were con-
ducted on the common more advanced oxazolidinones 7 and 8
derived from the rearranged products (vide infra).
To continue a synthesis towards 7 and 8, treatment of Over-
man's products 21 and 22 with p-TsOH in MeOH removed the
acetonide group and gave an inseparable mixture of diols 23 (92%,
Scheme 3). Their subsequent reaction with DBU resulted in pro-
duction of the desired cyclic carbamates 7 and 8 in 96% combined
yield. Column chromatography then allowed the straightforward
isolation of both diastereoisomers 7 and 8. On the other hand,
isothiocyanates 19 and 20 were initially converted into the cyclic
thiocarbamates 24 (94%) and 25 (88%) by the same conditions as
employed for the conversion of 21 and 22 to 23 (Scheme 3). The
replacement of the sulfur atom to oxygen in 24 and 25 was
accomplished with mesitylnitrile oxide in MeCN to afford scaffolds
7 and 8 in 89% and 92% yields, respectively.
At this stage, the relative C-4 stereochemistry of the common
products 7 and 8 was assigned by 1D NOESY experiments (Scheme
3). In the case of cyclic carbamate 8, the strong enhancements
between H-4 and H-5 protons indicate that these protons are in the
cis-relationship to each other. These findings demonstrate that the
C-4 carbon in 8 is (S)-configured. On the other hand, for derivative

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62
D. Jackova et al. / Carbohydrate Research 451 (2017) 59e71
Scheme 3. Reagents and conditions: (a) p-TsOH, MeOH, rt, 92% for 23; (b) DBU, CH2Cl2, rt, 96% (combined yield); (c) MNO, MeCN, rt, 89% for 7, 92% for 8.
Fig. 3. ORTEP structure of 24 showing the crystallographic numbering.
chain in CD3OD solution it was not possible to conduct the afore-
less active than its 5-epi-analogue 3 against all other evaluated
cancer cell lines, indicating that the configuration of the pyrrolidine
skeleton might be important for the biological activity. Iminosugar
4 was found to be more toxic on non-malignant mouse fibroblasts
NiH 3T3 than on screened cancer cell lines.
mentioned measurement on this sample. To allow comparisons, the
attached supplementary material includes 1H and 13C NMR data
(see Tables 1 and 2) for the pyrrolidine core of our final compounds
3 and 4 and known analogues reported in the literature [18].
2.2. Antiproliferative/cytotoxic activity
The final synthesized compounds 3 and 4 were evaluated for
in vitro antiproliferative/cytotoxic activities against seven human
cancer cell lines A-549 (non-small cell lung cancer), MCF-7
(mammary gland adenocarcinoma), MDA-MB-231 (mammary
gland adenocarcinoma), HTC-116 (human colon carcinoma), Caco-2
(human colon carcinoma), HeLa (cervical adenocarcinoma) and
Jurkat (acute T-lymphoblastic leukaemia); and a non-malignant cell
line NiH 3T3 (mouse fibroblasts) using the MTT assay [19]. The
anticancer agent cisplatin was included as positive control, and all
assays were carried out in triplicate. The obtained IC50 values
(Table 2) showed that targeted compound 3 displays higher anti-
proliferative/cytotoxic potencies against Caco-2 and Jurkat cancer
cell lines than cisplatin, whose IC50 values on these cells are at least
2.5e3 ꢂ higher. With the exception of HeLa cells, compound 4 was
3. Conclusions
In summary, we have accomplished the convergent synthesis of
two C-alkyl iminosugars 3 and 4. The key transformations of the
developed approach are synthesis of the common oxazolidinone
intermediates 7 and 8 using heterosigmatropic rearrangements to
establish the new C-N bond and Grubbs' cross metathesis chem-
istry followed by intramolecular cyclization to create the carbon
backbone and pyrrolidine function, respectively. The targeted
compound 3 displayed very good antiproliferative/cytotoxic activ-
ity against Caco-2 and Jurkat cancer cell lines. Its 5-epimer 4 was
found to be less potent on human cancer cell lines and more toxic
against non-malignant cells (NiH 3T3).

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D. Jackova et al. / Carbohydrate Research 451 (2017) 59e71
63
Table 1
content of the vessel was monitored 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 com-
pressed air. Melting points were recorded on a Kofler hot block, and
Crystal data and structure refinement parameters for compound 24.
24
Empirical formula
Formula weight
C22H25NO4S
399.50
are uncorrected. Small quantities of reagents (mL) were measured
Temperature, T (K)
293 (2)
with appropriate syringes (Hamilton). All reactions were per-
formed under an atmosphere of nitrogen, unless otherwise noted.
Wavelength,
l
(Å)
1.54184
Monoclinic
P21
Crystal system
Space group
Unit cell dimensions
4.1.1. 1,2-O-Isopropylidene-5-O-trityl-a-D-ribofuranose (12) [13]
IBX was added to a solution of 11 [12b] (10.8 g, 0.025 mol) in
MeCN (230 mL) and the resulting mixture was heated at reflux for
5 h. After cooling to room temperature, the solid parts were filtered
off, and the filtrate was concentrated in vacuo. Chromatography of
the residue on silica gel (n-hexane/ethyl acetate, 5:1) gave 10.0 g
a (Å)
b (Å)
7.07255 (13)
17.9943 (3)
8.92418 (19)
1083.54 (4)
2
b
¼ 107.438 (2)ꢀ
c (Å)
V (Å3)
Formula per unit cell, Z
Dcalcd (g/cm3)
1.224
1.542
424
Absorption coefficient,
F (0 0 0)
m
(mmꢁ1
)
(93%) of 1,2-O-isopropylidene-5-O-trityl-a-D-erythro-pentofur-
anos-3-ulose [13] as a white amorphous solid; [
a
]
26 þ123.5 (c 0.20,
D
Crystal size (mm)
0.86 ꢂ 0.58 ꢂ 0.12
5.195e75.716
ꢁ8 ꢃ h ꢃ 8
CHCl3), lit [13]. [
a]
D þ132 (c 4.7, CHCl3, temperature at 21e22 ꢀC). IR
q
Range for data collection (ꢀ)
(neat) ymax 1010, 1077, 1152, 1215, 1375, 1448, 1489, 1773 cmꢁ1; 1H
Index ranges
NMR (600 MHz, CDCl3):
d
1.47 (s, 6H, 2 ꢂ CH3), 3.32 (dd, 1H,
ꢁ22 ꢃ k ꢃ 22
ꢁ10 ꢃ l ꢃ 11
J ¼ 2.6 Hz, J ¼ 10.1 Hz, H-5), 3.50 (dd,1H, J ¼ 2.4 Hz, J ¼ 10.1 Hz, H-5),
4.41 (m, 1H, H-4), 4.54 (dd, 1H, J ¼ 1.0 Hz, J ¼ 4.5 Hz, H-2), 6.33 (d,
1H, J ¼ 4.5 Hz, H-1), 7.23e7.25 (m, 3H, Ph), 7.28e7.31 (m, 6H, Ph),
Independent reflections (Rint)
Completeness to 2
Reflections collected
Refinement method
4456 (0.0388)
98%
Q
¼ 74.98ꢀ
56522
7.34e7.36 (m, 6H, Ph); 13C NMR (100 MHz, CDCl3):
d 27.2 (CH3), 27.7
Full-matrix least-squares on F2
4456/250/253
1.058
Data/restraints/parameters
(CH3), 64.4 (C-5), 76.9 (C-2), 80.0 (C-4), 87.4 (Cq), 103.6 (C-1), 114.2
(Cq), 127.2 (3 ꢂ CHPh), 127.9 (6 ꢂ CHPh), 128.6 (6 ꢂ CHPh), 143.2
(3 ꢂ Ci), 210.2 (C-3). Anal. Calcd for C27H26O5: C, 75.33; H, 6.09.
Found: C, 75.20, H, 6.18.
To a solution of the obtained ulose [13] (9.9 g, 0.023 mol) in a
mixture of EtOH/CH2Cl2 (378 mL, 8:1) that had been pre-cooled to
0 ꢀC was added NaBH4 (1.74 g, 0.046 mol). After stirring at 0 ꢀC for
10 min and another 20 min at room temperature, the solvents were
evaporated, and the residue was partitioned between CH2Cl2
(290 mL) and brine (340 mL). The separated aqueous layer was then
washed with CH2Cl2 (2 ꢂ 290 mL). The combined organic extracts
were dried over Na2SO4, the solvent was evaporated, and the res-
idue was subjected to flash chromatography on silica gel (n-hexane/
Goodness-of-fit on F2
Final R indices [I > 2
R indices (all data)
Flack x parameter
s
(I)]
R1 ¼ 0.0415, wR2 ¼ 0.1263
R1 ¼ 0.0461, wR2 ¼ 0.1334
0.004 (8)
0.137 and ꢁ0.135
Largest diff. peak and hole (e/Åꢁ3
)
4. Experimental
4.1. Chemistry
All commercial reagents were used in the highest available
purity from Aldrich, Merck or Acros Organics without further pu-
rification. Solvents were dried and purified before use according to
standard procedures. For flash column chromatography on silica
gel, Kieselgel 60 (0.040e0.063 mm, 230e400 mesh, Merck) was
used. Solvents for flash chromatography (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
spectra were recorded in CDCl3, CD3OD and C6D6 on a Varian
Mercury Plus 400 FT NMR (400.13 MHz for 1H and 100.61 MHz for
13C) or on a Varian Premium COMPACT 600 (599.87 MHz for 1H and
150.84 MHz for 13C) spectrometer using TMS as internal reference.
ethyl acetate, 5:1) to afford 9.85 g (99%) of compound 12 as a white
26
foam; [
a
]
D
þ25.5 (c 0.20, CHCl3); lit [13]. [
a
]
þ25.8 (c 4.5, CHCl3,
D
temperature at 21e22 ꢀC). IR (neat) ymax 1011,1064,1114,1163,1213,
1373, 1447, 1490 cmꢁ1 1H NMR (400 MHz, CDCl3):
;
d
1.38 (s, 3H,
CH3), 1.57 (s, 3H, CH3), 2.31 (d, 1H, J ¼ 9.6 Hz, OH), 3.27 (dd, 1H,
J ¼ 4.8 Hz, J ¼ 10.4 Hz, H-5), 3.41 (dd, 1H, J ¼ 3.0 Hz, J ¼ 10.4 Hz, H-
5), 3.88e4.00 (m, 2H, H-3, H-4), 4.57e4.59 (m, 1H, H-2), 5.88 (d, 1H,
J ¼ 3.8 Hz, H-1), 7.21e7.26 (m, 3H, Ph), 7.27e7.32 (m, 6H, Ph),
7.44e7.48 (m, 6H, Ph); 13C NMR (100 MHz, CDCl3):
d 26.5 (CH3), 26.6
(CH3), 63.0 (C-5), 72.3 (C-3), 78.5(C-2), 79.8 (C-4), 86.7 (Cq), 104.2
(C-1), 112.6 (Cq), 127.0 (3 ꢂ CHPh), 127.8 (6 ꢂ CHPh), 128.7 (6 ꢂ CHPh),
143.8 (3 ꢂ Ci). Anal. Calcd for C27H28O5: C, 74.98; H, 6.53. Found: C,
74.82; H, 6.64.
For 1H,
¼ 0.0), CD3OD (
relative to CDCl3
d
are given in parts per million (ppm) relative to TMS
4.1.2. 1,2-O-Isopropylidene-a-D-ribofuranose (13) [14]
A solution of ribofuranose 12 (9.5 g, 0.022 mol) in CH2Cl2/MeOH
(96 mL, 2:1) was treated with CSA (0.256 g, 1.10 mmol) at room
temperature. After being stirred until no starting material was
observed on TLC (3 h), the reaction was quenched with Et3N
(0.2 mL), and solvents were evaporated. Purification of the residue
by flash chromatography on silica gel (n-hexane/ethyl acetate, 1:2)
provided 4.0 g (96%) of compound 13 as colourless crystals; mp
(
d
d
¼ 4.84 or
d
¼ 3.31), C6D6
(
d
¼ 7.15) and for 13
C
(d
¼ 77.00), CD3OD (
d
¼ 49.05) and C6D6
(
d
¼ 128.02). The multiplicity of the 13C NMR signals concerning the
13C-1H coupling was determined by the HSQC 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 (cmꢁ1).
Elemental analysis was performed on a Perkin-Elmer CHN 2400
elemental analyser. Optical rotations were measured on a P-2000
86e87 ꢀC; lit [14]. mp 85e86 ꢀC; [
[14]. [
D [21] þ42.7 (c 1.32, CHCl3). IR (neat) ymax 1018, 1087, 1115,
1142, 1172, 1215, 1240, 1370, 1380, 2886, 2421, 2956, 3232 cmꢁ1; 1H
NMR (400 MHz, CDCl3): 1.38 (s, 3H, CH3), 1.57 (s, 3H, CH3), 1.97 (br
a]
D [24] þ43.2 (c 0.91, CHCl3); lit
a
]
d
Jasco polarimeter and reported as follows: [a] (c in grams per
100 mL, solvent). Microwave reactions were carried out on the
focused microwave system (CEM Discover). The temperature
D
s,1H, OH), 2.46 (br s,1H, OH), 3.72e3.77 (m,1H, H-5), 3.82e3.86 (m,
1H, H-5), 3.94e4.04 (m, 2H, H-3, H-4), 4.59 (t, 1H, J ¼ 4.4 Hz, H-2),
5.83 (d, 1H, J ¼ 3.7 Hz, H-1); 13C NMR (100 MHz, CDCl3):
d 26.5

ꢀ
D. Jackova et al. / Carbohydrate Research 451 (2017) 59e71
64
Scheme 4. Reagents and conditions: (a) Boc2O, DMAP, Et3N, CH2Cl2, 0 ꢀC; (b) TsCl, Et3N, Me3N.HCl, CH2Cl2, rt; (c) Cs2CO3, EtOH, 0 ꢀC; (d) tridec-1-ene, Grubbs II, CH2Cl2, reflux; (e) (i)
TFA, CH2Cl2, 0 ꢀC; (ii) aq NH3, MeOH, rt; (f) H2, 20% Pd(OH)2/C, EtOAc/MeOH (1:3), 12 M HCl, rt.
Table 2
Antiproliferative activities of 3 and 4 on seven human cancer cell lines (A-549, MCF-7, MDA-MB-231, HCT-116, Caco-2, HeLa and Jurkat) and non-malignant mouse fibroblasts
NiH 3T3.
mol ꢂ Lꢁ1
)
a
Compd no.
Cell line, IC50
A-549
SD (
m
MCF-7
HCT-116
Caco-2
HeLa
NiH 3T3
MDA-MB-231
Jurkat
3
4
ND
23.8 6.8
9.5 0.2
22.0 4.11
26.6 3.22
15.6 0.3
9.3 3.29
30.9 3.92
15.3 0.5
5.1 0.87
25.9 0.56
15.2 0.3
22.4 8.83
18.4 1.43
13.1 0.2
ND
<10
20.87 0.3
24.6 1.99
28.4 0.89
17.5 0.5
5.8 1.41
ND
16.2 0.6
cisplatin
ND-not detected.
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 that decreased
amount of viable cells to 50% relative to untreated control cells, see Experimental part, section 4.2.2.).

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D. Jackova et al. / Carbohydrate Research 451 (2017) 59e71
65
(2 ꢂ CH3), 60.8 (C-5), 70.8 (C-3), 78.7 (C-2), 80.7 (C-4), 104.0 (C-1),
4.1.5. Ethyl (4S,5S,6R,2E)-5,7-bis(benzyloxy)-4,6-
(isopropylidenedioxy)hept-2-enoate (17)
112.8 (Cq). Anal. Calcd for C8H14O5: C, 50.52; H, 7.42. Found: C,
50.39; H, 7.51.
2,2-Dimethoxypropane (1.5 mL, 9.0 mmol) and p-TsOH.H2O
(57 mg, 0.3 mmol) were added to a solution of 16 (1.2 g, 3.0 mmol)
in dry CH2Cl2 (17 mL). After stirring at room temperature for
30 min, the whole mixture was washed with a saturated NaHCO3
solution (2 ꢂ 14 mL). The organic layer was dried over Na2SO4 and
concentrated. Chromatography of the residue on silica gel (n-hex-
ane/ethyl acetate, 11:1) furnished 1.28 g (97%) of compound 17 as a
4.1.3. 3,5-Di-O-benzyl-1,2-O-isopropylidene-a-D-ribofuranose (14)
[14]
To an ice-cold solution of 13 (3.25 g, 17.1 mmol) in dry DMF
(37 mL) were successively added NaH (1.97 g, 51.3 mmol, 60%
dispersion in mineral oil), BnBr (4.5 mL, 37.6 mmol) and TBAI
(0.189 g, 0.51 mmol). After stirring at 0 ꢀC for 10 min and another
50 min at room temperature, the excess hydride was decomposed
by addition of MeOH, and the resulting mixture was partitioned
between Et2O (200 mL) and cold water (370 mL). The separated
aqueous layer was then washed with another portion of Et2O
(200 mL). The combined organic extracts were dried over Na2SO4,
concentrated, and the residue was chromatographed on silica gel
(n-hexane/ethyl acetate, 7:1) to give 6.2 g (98%) of compound 14 as
colourless oil; [
a]
D
25 e39.0 (c 0.42, CHCl3). IR (neat) ymax 1092, 1660,
1716, 2868, 2991, 3030 cmꢁ1; 1H NMR (400 MHz, CDCl3):
d
1.30 (t,
3H, J ¼ 7.1 Hz, CH3), 1.49 (s, 3H, CH3), 1.50 (s, 3H, CH3), 3.31 (t, 1H,
J ¼ 9.7 Hz, H-5), 3.63 (dd, 1H, J ¼ 1.9 Hz, J ¼ 10.9 Hz, H-7), 3.72 (dd,
1H, J ¼ 4.2 Hz, J ¼ 10.9 Hz, H-7), 3.91 (ddd, 1H, J ¼ 1.9 Hz, J ¼ 4.2 Hz,
J ¼ 9.6 Hz, H-6), 4.21 (q, 2H, J ¼ 7.1 Hz, CH2), 4.34e4.37 (m, 1H, H-4),
4.39 (d, 1H, J ¼ 10.6 Hz, OCH2Ph), 4.47 (d, 1H, J ¼ 10.6 Hz, OCH2Ph),
4.55 (d, 1H, J ¼ 12.2 Hz, OCH2Ph), 4.66 (d, 1H, J ¼ 12.2 Hz, OCH2Ph),
6.16 (dd, 1H, J ¼ 1.5 Hz, J ¼ 15.7 Hz, H-2), 7.06 (dd, 1H, J ¼ 4.9 Hz,
a colourless oil; [
[21] þ86.6 (c 2.66, CHCl3). IR (neat) ymax 1018,1095,1125,1167,1213,
1308, 1371, 1453, 1496, 2864, 2985 cmꢁ1 1H NMR (400 MHz,
CDCl3):
a
]
D
[24] þ90.3 (c 0.64, CHCl3); lit [14]. [
a]
D
J ¼ 15.7 Hz, H-3), 7.16e7.18 (m, 2H, Ph), 7.25e7.39 (m, 8H, Ph); 13
C
NMR (100 MHz, CDCl3):
d 14.2 (CH3), 19.2 (CH3), 29.3 (CH3), 60.4
;
(CH2), 69.1 (C-7), 72.2 (C-4), 73.1 (C-6), 73.5 (OCH2Ph), 74.4 (C-5),
74.8 (OCH2Ph), 98.9 (Cq), 122.2 (C-2), 127.7 (CHPh), 128.0 (3 ꢂ CHPh),
128.2 (2 ꢂ CHPh), 128.4 (2 ꢂ CHPh), 128.5 (2 ꢂ CHPh), 137.1 (Ci), 138.0
(Ci), 144.4 (C-3), 166.3 (C¼O). Anal. Calcd for C26H32O6: C, 70.89; H,
7.32. Found: C, 71.00; H, 7.41.
d
1.36 (s, 3H, CH3), 1.60 (s, 3H, CH3), 3.56 (dd, 1H, J ¼ 3.9 Hz,
J ¼ 11.2 Hz, H-5), 3.76 (dd, 1H, J ¼ 2.0 Hz, J ¼ 11.2 Hz, H-5), 3.86 (dd,
1H, J ¼ 4.4 Hz, J ¼ 9.1 Hz, H-3), 4.16e4.19 (m, 1H, H-4), 4.48e4.58
(m, 4H, H-2, OCH2Ph, H-OCH2Ph), 4.73 (d, 1H, J ¼ 12.0 Hz, OCH2Ph),
5.75 (d, 1H, J ¼ 3.7 Hz, H-1), 7.25e7.36 (m, 10H, Ph); 13C NMR
(100 MHz, CDCl3):
d 26.5 (CH3), 26.8 (CH3), 67.9 (C-5), 72.2
4.1.6. (4S,5S,6R,2E)-5,7-Bis(benzyloxy)-4,6-(isopropylidenedioxy)
hept-2-en-1-ol (18) [14]
(OCH2Ph), 73.5 (OCH2Ph), 77.1 (C-3), 77.4 (C-2), 77.9 (C-4), 104.1 (C-
1), 112.9 (Cq), 127.6 (CHPh), 127.7 (2 ꢂ CHPh), 128.0 (3 ꢂ CHPh), 128.3
(2 ꢂ CHPh), 128.4 (2 ꢂ CHPh), 137.6 (Ci), 138.0 (Ci). Anal. Calcd for
DIBAl-H (6.8 mL, 8.16 mmol, ~1.2 M solution in toluene) was
added dropwise to a solution of ester 17 (1.2 g, 2.72 mmol) that had
been pre-cooled to ꢁ30 ꢀC. After being stirred at the same tem-
perature until no starting material was observed on TLC (30 min),
the reaction was quenched with MeOH (1.6 mL), and the whole
mixture was treated with a 30% K/Na tartrate solution (43 mL) with
vigorous stirring over 1 h at room temperature. The separated
aqueous layer was extracted with CH2Cl2 (2 ꢂ 25 mL). The com-
bined organic extracts were dried over Na2SO4, concentrated, and
the residue was purified by column chromatography on silica gel
(n-hexane/ethyl acetate, 2:1) to give 1.04 g (96%) of allylic alcohol
C
22H26O5: C, 71.33; H, 7.07. Found: C, 71.25; H, 7.20.
4.1.4. Ethyl (4S,5S,6R,2E)-5,7-bis(benzyloxy)-4,6-dihydroxyhept-2-
enoate (16)
Compound 14 (6.1 g, 16.5 mmol) was treated with a solution of
80% aq TFA (29 mL) at room temperature. After being stirred until
no starting material was observed (judged by TLC, 2 h), the mixture
was diluted with EtOAc (40 mL) and water (40 mL), and neutralized
with the solid Na2CO3. The separated aqueous layer was washed
with EtOAc (4 ꢂ 100 mL). The combined organic extracts were dried
over Na2SO4, the filtrate was concentrated in vacuo, and the residue
was subjected to flash chromatography on silica gel (n-hexane/
ethyl acetate, 1:1) to afford 4.35 g (80%) of compound 15 [14] as a
white amorphous solid that was used immediately in the next re-
action without spectral characterization.
Ylide, Ph3P ¼ CHCO2Et, (2.85 g, 8.17 mmol) and benzoic acid
(55.4 mg, 0.454 mmol) were successively added to a solution of 15
(1.5 g, 4.54 mmol) in dry CH2Cl2 (30 mL) at room temperature. After
stirring for 8 h, the solvent was removed in vacuo, and the crude
product was chromatographed through a short silica gel column (n-
hexane/ethyl acetate, 2:1, then CH2Cl2/ethyl acetate, 10:1) to afford
18 as white crystals; mp 89e90 ꢀC; lit [14]. mp 92e93 ꢀC; [
[24] þ5.2 (c 0.62, CHCl3); lit [14]. [ [24] þ9.1 (c 1.22, CHCl3). IR
(neat) ymax 1011, 1093, 2865, 2992, 3477 cmꢁ1; 1H NMR (400 MHz,
CDCl3): 1.28 (br s, 1H, OH), 1.47 (s, 3H, CH3), 1.51 (s, 3H, CH3), 3.30
a]
D
a]
D
d
(t,1H, J ¼ 9.6 Hz, H-5), 3.65 (dd, 1H, J ¼ 1.9 Hz, J ¼ 10.9 Hz, H-7), 3.73
(dd, 1H, J ¼ 4.3 Hz, J ¼ 10.9 Hz, H-7), 3.90 (ddd, 1H, J ¼ 1.9 Hz,
J ¼ 4.1 Hz, J ¼ 9.6 Hz, H-6), 4.10e4.16 (m, 2H, 2 ꢂ H-1), 4.22 (dd, 1H,
J ¼ 7.2 Hz, J ¼ 9.1 Hz, H-4), 4.39 (d, 1H, J ¼ 10.8 Hz, OCH2Ph), 4.48 (d,
1H, J ¼ 10.8 Hz, OCH2Ph), 4.56 (d, 1H, J ¼ 12.3 Hz, OCH2Ph), 4.68 (d,
1H, J ¼ 12.3 Hz, OCH2Ph), 5.72 (dd, 1H, J ¼ 6.9 Hz, J ¼ 15.5 Hz, H-3),
6.01 (td, 1H, J ¼ 5.2 Hz, J ¼ 15.5 Hz, H-2) 7.15e7.17 (m, 2H, Ph),
7.27e7.38 (m, 8H, Ph); 13C NMR (100 MHz, CDCl3):
d 19.4 (CH3), 29.5
1.22 g (67%) of compound 16 as a colourless oil; [
0.22, CHCl3). IR (neat) ymax 1071, 1267, 1655, 1701, 2867, 2901 cmꢁ1
1H NMR (400 MHz, CDCl3):
a
]
[24] ꢁ11.8 (c
(CH3), 63.0 (C-1), 69.3 (C-7), 73.0 (C-6), 73.5 (OCH2Ph), 73.7 (C-4),
74.4 (OCH2Ph, C-5), 98.7 (Cq), 127.7 (CHPh), 127.9 (CHPh), 128.0
(2 ꢂ CHPh), 128.3 (2 ꢂ CHPh), 128.4 (4 ꢂ CHPh), 128.8 (C-3), 133.4 (C-
2), 137.7 (Ci), 138.1 (Ci). Anal. Calcd for C24H30O5: C, 72.34; H, 7.59.
Found: C, 72.25; H, 7.67.
There are some differences in the coupling constants of the vinyl
protons H-2 and H-3 between our compound 18 and the sample
reported by Xie and co-workers [14]. Chemical shifts of these
protons are in good agreement with those published in the litera-
ture [14].
D
;
d
1.29 (t, 3H, J ¼ 7.1 Hz, CH3), 2.71 (d, 1H,
J ¼ 5.9 Hz, OH), 3.22 (d, 1H, J ¼ 4.8 Hz, OH), 3.53 (dd, 1H, J ¼ 5.6 Hz,
J ¼ 7.3 Hz, H-5), 3.60 (dd, 1H, J ¼ 5.3 Hz, J ¼ 9.6 Hz, H-7), 3.67 (dd,
1H, J ¼ 3.7 Hz, J ¼ 9.6 Hz, H-7), 3.88e3.93 (m, 1H, H-6), 4.20 (q, 2H,
J ¼ 7.1 Hz, CH2), 4.49e4.62 (m, 5H, H-4, 2 ꢂ OCH2Ph), 6.14 (dd, 1H,
J ¼ 1.8 Hz, J ¼ 15.7 Hz, H-2), 7.11 (dd,1H, J ¼ 4.7 Hz, J ¼ 15.7 Hz, H-3),
7.24e7.39 (m, 10H, Ph); 13C NMR (100 MHz, CDCl3):
d 14.3 (CH3),
60.4 (CH2), 70.6 (C-7), 71.7 (C-6), 72.2 (C-4), 73.5 (OCH2Ph), 74.1
(OCH2Ph), 81.3 (C-5), 121.7 (C-2), 128.0 (3 ꢂ CHPh), 128.1 (CHPh),
128.2 (2 ꢂ CHPh), 128.5 (4 ꢂ CHPh), 137.5 (2 ꢂ Ci), 146.5 (C-3), 166.4
(C¼O). Anal. Calcd for C23H28O6: C, 69.98; H, 7.05. Found: C, 69.86;
H, 6.96.
4.1.7. (4R,5S,6S)-5-(Benzyloxy)-4-[(benzyloxy)methyl]-2,2-
dimethyl-6-[(E)-30-thiocyanatoprop-10-en-10-yl)-1,3-dioxane] (9)
To an ice-cold solution of alcohol 18 (0.667 g, 1.7 mmol) in dry

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D. Jackova et al. / Carbohydrate Research 451 (2017) 59e71
66
CH2Cl2 (14 mL) were successively added Et3N (0.48 mL, 3.4 mmol)
and MsCl (0.26 mL, 3.4 mmol). After being stirred at 0 ꢀC for 10 min
and another 20 min at room temperature, the mixture wad diluted
with Et2O, the solid part was filtered off, and the filtrate was
concentrated in vacuo. The obtained residue was used immediately
in the next reaction without purification.
KSCN (0.30 g, 3.07 mmol) was added to a solution of the crude
mesylate (0.81 g, 1.70 mmol) in MeCN (14 mL) at 0 ꢀC. After the
mixture had stirred for 24 h at room temperature, the resulting
yellow mixture was diluted with Et2O. The insoluble material was
removed by filtration and washed with Et2O. After the solvent was
evaporated, the residue was flash-chromatographed on silica gel
(n-hexane/ethyl acetate, 9:1) to furnish 0.684 g (93%) of compound
1H, H-30), 5.87 (ddd, 1H, J ¼ 6.3 Hz, J ¼ 10.2 Hz, J ¼ 16.8 Hz, H-20),
7.19e7.21 (m, 2H, Ph), 7.28e7.41 (m, 8H, Ph); 13C NMR (150 MHz,
CDCl3):
d
19.2 (CH3), 29.2 (CH3), 60.0 (C-10), 69.4 (C-100), 71.4 (C-5),
73.3 (C-4), 73.7 (OCH2Ph), 74.7 (OCH2Ph), 74.9 (C-6), 99.2 (Cq), 118.2
(C-30), 127.7 (CHPh), 128.0 (2 ꢂ CHPh), 128.1 (2 ꢂ CHPh), 128.2 (CHPh),
128.4 (2 ꢂ CHPh), 128.6 (2 ꢂ CHPh), 133.0 (C-20), 136.0 (NCS), 137.3
(Ci), 138.1 (Ci). Anal. Calcd for C25H29NO4S: C, 68.31; H, 6.65; N, 3.19.
Found: C, 68.25; H, 6.73; N, 3.26.
Diastereoisomer 20: colourless oil; [
(neat) ymax 1090, 1202, 1381, 2040, 2869, 2992 cmꢁ1
(600 MHz, CDCl3): 1.47 (s, 3H, CH3), 1.50 (s, 3H, CH3), 3.44 (t, 1H,
a]
D
26 e91.9 (c 0.58, CHCl3). IR
;
1H NMR
d
J ¼ 9.6 Hz, H-5), 3.64 (dd, 1H, J ¼ 2.2 Hz, J ¼ 11.1 Hz, H-100), 3.74 (dd,
1H, J ¼ 4.0 Hz, J ¼ 11.1 Hz, H-100), 3.84e3.87 (m, 1H, H-4), 3.99 (dd,
1H, J ¼ 2.4 Hz, J ¼ 9.7 Hz, H-6), 4.36e4.38 (m, 1H, H-10), 4.42 (d, 1H,
J ¼ 11.0 Hz, OCH2Ph), 4.44 (d, 1H, J ¼ 11.0 Hz, OCH2Ph), 4.56 (d, 1H,
J ¼ 12.2 Hz, OCH2Ph), 4.69 (d, 1H, J ¼ 12.2 Hz, OCH2Ph), 5.23e5.26
(m, 1H, H-30), 5.30 (d, 1H, J ¼ 10.2 Hz, H-30), 5.91 (ddd, 1H, J ¼ 7.1 Hz,
J ¼ 10.2 Hz, J ¼ 17.2 Hz, H-20), 7.14e7.16 (m, 2H, Ph), 7.27e7.37 (m,
9 as a colourless oil; [
970, 1093, 1201, 2153, 2865, 2992, 3029 cmꢁ1
CDCl3):
a
]
[23] ꢁ4.2 (c 0.38, CHCl3). IR (neat) ymax
D
;
1H NMR (400 MHz,
d
1.48 (s, 3H, CH3), 1.51 (s, 3H, CH3), 3.31 (t, 1H, J ¼ 9.6 Hz, H-
5), 3.50e3.59 (m, 2H, 2 ꢂ H-30), 3.63 (dd, 1H, J ¼ 2.0 Hz, J ¼ 10.9 Hz,
H-100), 3.71 (dd, 1H, J ¼ 4.3 Hz, J ¼ 10.9 Hz, H-100), 3.90 (ddd, 1H,
J ¼ 2.0 Hz, J ¼ 4.3 Hz, J ¼ 9.6 Hz, H-4), 4.24 (dd, 1H, J ¼ 4.9 Hz,
J ¼ 9.5 Hz, H-6), 4.43 (d, 1H, J ¼ 10.9 Hz, OCH2Ph), 4.56 (d, 1H,
J ¼ 12.3 Hz, OCH2Ph), 4.58 (d, 1H, J ¼ 10.9 Hz, OCH2Ph), 4.66 (d, 1H,
J ¼ 12.3 Hz, OCH2Ph), 5.90 (dd, 1H, J ¼ 4.9 Hz, J ¼ 15.3 Hz, H-10), 5.97
(dd, 1H, J ¼ 6.6 Hz, J ¼ 15.3 Hz, H-20), 7.17e7.38 (m, 10H, Ph); 13C
4H, Ph); 13C NMR (150 MHz, CDCl3):
d 19.1 (CH3), 29.1 (CH3), 61.5 (C-
10), 69.2 (C-100), 71.0 (C-5), 73.5 (C-4), 73.6 (OCH2Ph), 73.8 (OCH2Ph),
75.2 (C-6), 99.2 (Cq), 119.4 (C-30), 127.7 (CHPh), 127.8 (2 ꢂ CHPh),
128.0 (3 ꢂ CHPh), 128.4 (2 ꢂ CHPh), 128.5 (2 ꢂ CHPh), 131.4 (C-20),
136.3 (NCS), 137.4 (Ci), 138.0 (Ci). Anal. Calcd for C25H29NO4S: C,
68.31; H, 6.65; N, 3.19. Found: C, 68.22; H, 6.71; N, 3.25.
NMR (100 MHz, CDCl3): d
19.3 (CH3), 29.4 (CH3), 35.9 (C-30), 69.2 (C-
100), 72.7 (C-6), 73.1 (C-4), 73.5 (OCH2Ph), 74.5 (OCH2Ph), 74.6 (C-5),
98.8 (Cq), 111.9 (SCN), 124.9 (C-20), 127.7 (CHPh), 127.9 (CHPh), 128.0
(2 ꢂ CHPh),128.1 (2 ꢂ CHPh),128.4 (4 ꢂ CHPh),135.0 (C-10),137.6 (Ci),
138.1 (Ci). Anal. Calcd for C25H29NO4S: C, 68.31; H, 6.65; N, 3.19.
Found: C, 68.22; H, 6.73; N, 3.23.
4.1.9. N-[(R)-1-{(40S,50S,60R)-5'-(Benzyloxy]-6'-[(benzyloxy)
methyl]-20,20-dimethyl-10,3-dioxan-40-yl}allyl]-2,2,2-
trichloroacetamide (21) and N-[(S)-1-{(40S,50S,60R)-5'-(benzyloxy]-
6'-[(benzyloxy)methyl]-20,20-dimethyl-10,3-dioxan-40-yl}allyl]-2,2,2-
trichloroacetamide (22)
4.1.8. (4R,5S,6S)-5-(Benzyloxy)-4-[(benzyloxy)methyl]-6-[(R)-10-
isothiocyanatoallyl]-2,2-dimethyl-1,3-dioxane (19) and (4R,5S,6S)-
5-(benzyloxy)-4-[(benzyloxy)methyl]-6-[(S)-10-
To an ice-cold solution of 18 (85 mg, 0.21 mmol) in dry CH2Cl2
(1.1 mL) was added DBU (3.2
mL, 0.021 mmol) followed by tri-
chloroacetonitrile (42.7
m
L 0.42 mmol). After being stirred at 0 ꢀC
isothiocyanatoallyl]-2,2-dimethyl-1,3-dioxane (20)
for 10 min and then for a further 20 min at room temperature, the
mixture was filtered through a small pad of silica gel. Removal of
the solvent provided the crude imidate 10 that was used in the next
reaction without purification.
4.1.8.1. Conventional procedure. A solution of thiocyanate 9 (86 mg,
0.196 mmol) in dry n-heptane (1.7 mL) was heated at 90 ꢀC for 6 h
under a nitrogen atmosphere before cooling to room temperature.
After the solvent was evaporated, the residue was purified by flash
chromatography on silica gel (n-hexane/ethyl acetate, 17:1, then
9:1) to give a diastereomeric mixture of the corresponding iso-
thiocyanates 19 and 20 (52 mg, 60%).
Imidate 10 (0.115 g, 0.21 mmol) was weighed to a 10-mL glass
pressure microwave tube equipped with a magnetic stirbar. o-
Xylene (2.9 mL) followed by anhydrous K2CO3 (33.6 mg, 0.24 mmol)
were added, the tube was closed with a silicone septum, and the
reaction was subjected to microwave irradiation at 150 ꢀC (3.5 h) or
170 ꢀC (1 h). After cooling to room temperature, the insoluble parts
were filtered off, and the filtrate was concentrated in vacuo. Puri-
fication of the residue on silica gel (n-hexane/ethyl acetate, 9:1)
afforded an inseparable mixture of diastereoisomers 21 and 22
(98 mg, 85% for 150 ꢀC; 0.108 g, 94% for 170 ꢀC).
4.1.8.2. Microwave-assisted synthesis. Thiocyanate
9
(86 mg,
0.196 mmol) was weighed in a 10-mL glass pressure microwave
tube equipped with a magnetic stirbar. n-Heptane (1.7 mL) was
added, the tube was closed with a silicon septum, and the resulting
mixture was subjected to microwave irradiation at 90 ꢀC for 1 h.
The mixture was allowed to cool to room temperature, the solvent
was evaporated, and the residue was purified by flash chromatog-
raphy on silica gel (n-hexane/ethyl acetate, 17:1, then 9:1) to afford
a mixture of isothiocyanates 19 and 20 (66 mg, 77%).
Requiring a greater amount of the rearranged products 21 and
22, the procedure was repeated at 170 ꢀC with 1 g of the starting
imidate 10 using a big vessel.
Requiring a greater amount of the pure rearranged products 19
and 20, they were obtained on a multi-gram scale by the
microwave-assisted method in n-heptane at 90 ꢀC using a big
vessel. Column chromatography allowed the isolation of both ste-
reoisomers in pure form.
4.1.10. (4R,5S)-5-[(10R,20R)-10,30-Bis(benzyloxy)-20-hydroxypropyl]-
4-vinyloxazolidin-2-one (7) and (4S,5S)-5-[(10R,20R)-10,30-
bis(benzyloxy)-20-hydroxypropyl]-4-vinyloxazolidin-2-one (8)
p-TsOH.H2O (16.7 mg, 0.088 mmol) was added to a solution of a
mixture of trichloroacetamides 21 and 22 (0.24 g, 0.44 mmol) in
MeOH (8.7 mL). After stirring at room temperature for 22 h, the
solvent was evaporated, and the residue was subjected to flash
chromatography through a short silica gel column (n-hexane/ethyl
acetate, 3:1) to yield 0.205 g (92%) of an inseparable mixture of
diols 23.
Diastereoisomer 19: colourless oil; [
CHCl3). IR (neat) ymax 1064, 1202, 1361, 2050, 2868, 2924 cmꢁ1; 1H
NMR (600 MHz, CDCl3): 1.43 (s, 3H, CH3), 1.47 (s, 3H, CH3), 3.60 (t,
a]
[21] ꢁ28.6 (c 0.28,
D
d
1H, J ¼ 9.5 Hz, H-5), 3.67 (dd, 1H, J ¼ 2.1 Hz, J ¼ 11.2 Hz, H-100), 3.71
(dd, 1H, J ¼ 2.0 Hz, J ¼ 9.5 Hz, H-6), 3.75 (dd, 1H, J ¼ 4.4 Hz,
J ¼ 11.2 Hz, H-100), 3.88 (ddd, 1H, J ¼ 2.1 Hz, J ¼ 4.5 Hz, J ¼ 9.5 Hz, H-
4), 4.30e4.32 (m, 1H, H-10), 4.51 (d, 1H, J ¼ 11.0 Hz, OCH2Ph), 4.56
(d, 1H, J ¼ 11.0 Hz, OCH2Ph), 4.59 (d, 1H, J ¼ 12.2 Hz, OCH2Ph), 4.72
(d, 1H, J ¼ 12.2 Hz, OCH2Ph), 5.25e5.27 (m, 1H, H-30), 5.32e5.35 (m,
4.1.10.1. From diols 23. DBU (6 mL, 38.0 mmol) was added to a so-
lution of 23 (0.191 g, 0.38 mmol) in dry CH2Cl2 (4.4 mL) at room
temperature. After stirring for 28 h, the solvent was evaporated,

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D. Jackova et al. / Carbohydrate Research 451 (2017) 59e71
67
and the residue was purified by flash chromatography (n-hexane/
ethyl acetate, 1:1) to afford both cyclic carbamates 7 and 8 in
combined yield of 96% (0.14 g). Repeated chromatography gave the
pure oxazolidinones 7 and 8 as colourless oils.
(OCH2Ph), 74.9 (OCH2Ph), 77.8 (C-10), 88.5 (C-5), 119.3 (C-200), 128.0
(2 ꢂ CHPh), 128.1 (2 ꢂ CHPh), 128.5 (2 ꢂ CHPh), 128.6 (4 ꢂ CHPh),
134.7 (C-100), 137.1 (Ci), 137.2 (Ci), 188.9 (C-2). Anal. Calcd for
C22H25NO4S: C, 66.14; H, 6.31; N, 3.51. Found: C, 66.20; H, 6.27; N,
3.60.
4.1.10.2. From
thiocarbamates
24
and
25,
respectively.
Modification of 24 into 7: Mesitylnitrile oxide (48.4 mg, 0.30 mmol)
was added to a solution of 24 (0.10 g, 0.25 mmol) in MeCN (2.4 mL)
at room temperature. After stirring for 30 min, the solvent was
removed in vacuo, and the residue was chromatographed on silica
gel (n-hexane/ethyl acetate,1:1) to give 85 mg (89%) of carbamate 7.
Modification of 25 into 8: According to the same procedure
described for the preparation of 7, thiocarbamate 25 (80 mg,
0.20 mmol) and MNO (38.7 mg, 0.24 mmol) provided after flash
chromatography on silica gel (n-hexane/ethyl acetate, 1:1) 71 mg
(92%) of derivative 8.
4.1.12. (4S,5S)-5-[(10R,20R)-10,30-Bis(benzyloxy)-20-hydroxypropyl]-
4-vinyloxazolidine-2-thione (25)
By the same procedure as described for the preparation of thi-
ocarbamate 24, compound 20 (0.20 g, 0.455 mmol) and p-TsOH
(17.3 mg, 0.091 mmol) afforded after flash chromatography on silica
gel (n-hexane/ethyl acetate, 2:1) 0.16 g (88%) of compound 25 as
white crystals; mp 114e116 ꢀC; [
a]
[21] ꢁ17.4 (c 0.35, CHCl3). IR
D
(neat) ymax 1082, 1156, 1455, 1511, 1734, 3275, 3567 cmꢁ1; 1H NMR
(400 MHz, CDCl3):
d
2.81 (s, 1H, OH), 3.57e3.64 (m, 2H, 2 ꢂ H-30),
3.95 (t, 1H, J ¼ 5.9 Hz, H-10), 4.05e4.11 (m, 1H, H-20), 4.48 (d, 1H,
J ¼ 11.8 Hz, OCH2Ph), 4.52e4.55 (m, 2H, H-OCH2Ph, H-4), 4.61 (d,
1H, J ¼ 11.0 Hz, OCH2Ph), 4.67 (d, 1H, J ¼ 11.0 Hz, OCH2Ph), 5.10 (dd,
1H, J ¼ 5.7 Hz, J ¼ 8.8 Hz, H-5), 5.25 (d, 1H, J ¼ 10.2 Hz, H-200), 5.30
(d, 1H, J ¼ 17.2 Hz, H-200), 5.95e6.04 (m, 1H, H-100), 7.23e7.37 (m,
Diastereoisomer 7: [
a
]
D [21] þ72.8 (c 0.50, CHCl3). IR (neat) ymax
1088,1227,1454,1736, 2864, 3292 cmꢁ1; 1H NMR (400 MHz, CDCl3):
d
2.73 (d, 1H, J ¼ 5.7 Hz, OH), 3.53 (dd, 1H, J ¼ 5.5 Hz, J ¼ 9.5 Hz, H-
30), 3.61 (dd,1H, J ¼ 3.1 Hz, J ¼ 9.5 Hz, H-30), 3.72e3.78 (m,1H, H-20),
3.85 (dd, 1H, J ¼ 2.7 Hz, J ¼ 8.2 Hz, H-10), 4.39 (t, 1H, J ¼ 6.6 Hz, H-4),
4.46e4.53 (m, 3H, OCH2Ph, H-OCH2Ph), 4.67 (dd, 1H, J ¼ 2.7 Hz,
J ¼ 6.2 Hz, H-5), 4.75 (d, 1H, J ¼ 11.0 Hz, OCH2Ph), 5.18 (d, 1H,
J ¼ 10.2 Hz, H-200), 5.25 (d, 1H, J ¼ 17.1 Hz, H-200), 5.82 (ddd, 1H,
J ¼ 7.0 Hz, J ¼ 10.2 Hz, J ¼ 17.1 Hz, H-100), 5.96 (br s, 1H, NH),
10H, Ph), 7.83 (s, 1H, NH); 13C NMR (100 MHz, CDCl3):
d 61.2 (C-4),
70.0 (C-20), 70.2 (C-30), 73.4 (OCH2Ph), 74.2 (OCH2Ph), 77.8 (C-10),
84.2 (C-5), 120.4 (C-200), 127.8 (CHPh), 127.9 (5 ꢂ CHPh), 128.3
(2 ꢂ CHPh), 128.6 (2 ꢂ CHPh), 132.3 (C-100), 137.4 (Ci), 137.5 (Ci), 188.6
(C-2). Anal. Calcd for C22H25NO4S: C, 66.14; H, 6.31; N, 3.51. Found:
C, 66.05; H, 6.36; N, 3.43.
7.22e7.36 (m, 10H, Ph); 13C NMR (100 MHz, CDCl3):
d 54.9 (C-4),
69.1 (C-20), 70.5 (C-30), 73.4 (OCH2Ph), 74.8 (OCH2Ph), 78.2 (C-10),
82.2 (C-5), 117.7 (C-200) 127.9 (3 ꢂ CHPh), 128.0 (CHPh), 128.3
(2 ꢂ CHPh), 128.4 (2 ꢂ CHPh), 128.5 (2 ꢂ CHPh), 136.5 (C-100), 137.4
(2 ꢂ Ci), 158.8 (C-2). Anal. Calcd for C22H25NO5: C, 68.91; H, 6.57; N,
3.65. Found: C, 68.85; H, 6.49; N, 3.58.
4.1.13. tert-Butyl (4R,5S)-5-[(10R,20R)-10,30-bis(benzyloxy)-20-
hydroxypropyl]-2-oxo-4-vinyloxazolidine-3-carboxylate (26)
Boc2O (0.116 g, 0.532 mmol), Et3N (53.4
mL, 0.38 mmol) and
DMAP (93 mg, 0.76 mmol) were successively added to a solution of
oxazolidinone 7 (0.146 g, 0.38 mmol) in CH2Cl2 (10 mL) that had
been pre-cooled to 0 ꢀC. After stirring at the same temperature for
10 min, the mixture was poured into a saturated NH4Cl solution
(15 mL). The separated aqueous phase was then extracted with
CH2Cl2 (3 ꢂ 10 mL). The combined organic layers were dried over
Na2SO4, the solvent was evaporated, and the residue was subjected
to flash chromatography on silica gel (n-hexane/ethyl acetate, 2:1)
to give 0.143 g (78%) of compound 26 as a white solid; mp
Diastereoisomer 8: [
a
]
[21] ꢁ7.9 (c 0.48, CHCl3). IR (neat) ymax
D
1067, 1378, 1454, 1746, 2856, 2923, 3285 cmꢁ1; 1H NMR (400 MHz,
CDCl3):
d
2.92 (d, 1H, J ¼ 4.2 Hz, OH), 3.62e3.66 (m, 2H, 2 ꢂ H-30),
3.85 (dd, 1H, J ¼ 4.7 Hz, J ¼ 7.2 Hz, H-10), 4.12e4.18 (m, 1H, H-20),
4.34 (t, 1H, J ¼ 7.7 Hz, H-4), 4.48e4.55 (m, 2H, OCH2Ph), 4.57 (d, 1H,
J ¼ 11.1 Hz, OCH2Ph), 4.71 (d, 1H, J ¼ 11.1 Hz, OCH2Ph), 4.78 (t, 1H,
J ¼ 7.6 Hz, H-5), 5.23e5.28 (m, 2H, H-200), 5.95 (ddd, 1H, J ¼ 7.5 Hz,
J ¼ 10.2 Hz, J ¼ 17.4 Hz, H-100), 6.13 (br s, 1H, NH), 7.23e7.36 (m, 10H,
Ph); 13C NMR (100 MHz, CDCl3):
d
57.4 (C-4), 70.4 (C-20, C-30), 73.3
91e92 ꢀC; [
1311, 1732, 1759, 2923, 2976, 3443 cmꢁ1
CDCl3):
a
]
D [21] þ67.1 (c 0.69, CHCl3). IR (neat) ymax 1088, 1146,
(OCH2Ph), 73.6 (OCH2Ph), 77.7 (C-10), 78.1 (C-5), 119.0 (C-200), 127.7
(3 ꢂ CHPh), 127.8 (3 ꢂ CHPh), 128.3 (2 ꢂ CHPh), 128.4 (2 ꢂ CHPh),
133.7 (C-100), 137.6 (Ci), 137.7 (Ci), 158.8 (C-2). Anal. Calcd for
;
1H NMR (400 MHz,
d
1.45 (s, 9H, 3 ꢂ CH3), 2.41 (d, 1H, J ¼ 5.6 Hz, OH), 3.55 (dd,
1H, J ¼ 4.7 Hz, J ¼ 9.5 Hz, H-30), 3.61 (dd,1H, J ¼ 2.9 Hz, J ¼ 9.5 Hz, H-
30), 3.75e3.83 (m, 2H, H-20, H-10), 4.45 (d, 1H, J ¼ 10.7 Hz, OCH2Ph),
4.50 (d, 1H, J ¼ 11.7 Hz, OCH2Ph), 4.55 (d, 1H, J ¼ 11.7 Hz, OCH2Ph),
4.60 (dd, 1H, J ¼ 2.1 Hz, J ¼ 3.7 Hz, H-5), 4.69 (d, 1H, J ¼ 10.7 Hz,
OCH2Ph), 4.75 (dd, 1H, J ¼ 3.7 Hz, J ¼ 7.1 Hz, H-4), 5.26e5.30 (m, 2H,
2 ꢂ H-200), 5.87 (ddd, 1H, J ¼ 7.1 Hz, J ¼ 10.3 Hz, J ¼ 17.3 Hz, H-100),
C
22H25NO5: C, 68.91; H, 6.57; N, 3.65. Found: C, 68.82; H, 6.44; N,
3.56.
4.1.11. (4R,5S)-5-[(10R,20R)-10,30-Bis(benzyloxy)-20-hydroxypropyl]-
4-vinyloxazolidine-2-thione (24)
A solution of isothiocyanate 19 (0.15 g, 0.34 mmol) in MeOH
(4.4 mL) was treated with p-TsOH (12.9 mg, 0.068 mmol). After
being stirred at room temperature until no starting material was
observed (judged by TLC, 24 h), the mixture was quenched with
Et3N, the solvent was evaporated, and the residue was flash-
chromatographed on silica gel (n-hexane/ethyl acetate, 3:1) to
furnish 0.128 g (94%) of compound 24 as colourless crystals; mp
7.18e7.40 (m, 10H, Ph); 13C NMR (100 MHz, CDCl3):
d
27.8 (3 ꢂ CH3),
57.1 (C-4), 69.1 (C-20), 70.2 (C-30), 73.5 (OCH2Ph), 75.0 (OCH2Ph),
78.6 (C-10), 78.8 (C-5), 83.6 (Cq), 117.9 (C-200), 128.0 (2 ꢂ CHPh), 128.1
(2 ꢂ CHPh), 128.4 (4 ꢂ CHPh), 128.6 (2 ꢂ CHPh), 135.2 (C-100), 137.0
(Ci),137.3 (Ci),149.0 (C¼O),152.1 (C-2). Anal. Calcd for C27H33NO7: C,
67.06; H, 6.88; N, 2.90. Found: C, 67.16; H, 6.79; N, 2.96.
During this reaction we also isolated 22 mg (10%) of tert-butyl
(4R,5S)-5-{(10S,20R)-10,30-bis(benzyloxy)-2'-[(tert-butoxycarbonyl)
oxy]propyl}-2-oxo-4-vinyloxazolidine-3-carboxylate as a colour-
107e109 ꢀC; [
1249, 1374, 1497, 2861, 3249, 3589 cmꢁ1
CDCl3):
a
]
[20] þ87.0 (c 0.48, CHCl3). IR (neat) ymax 1091,
D
;
1H NMR (400 MHz,
d
2.41 (d, 1H, J ¼ 6.2 Hz, OH), 3.55 (dd, 1H, J ¼ 5.1 Hz,
less oil; [
1252, 1274, 1368, 1742, 1796, 1817, 2870, 2932, 2979 cmꢁ1; 1H NMR
(400 MHz, CDCl3):
a
]
[21] þ18.2 (c 0.78, CHCl3). IR (neat) ymax 1066, 1156,
D
J ¼ 9.4 Hz, H-30), 3.61 (dd, 1H, J ¼ 3.2 Hz, J ¼ 9.4 Hz, H-30), 3.65e3.71
(m, 1H, H-20), 3.97 (dd, 1H, J ¼ 2.1 Hz, J ¼ 8.6 Hz, H-10), 4.45e4.55
(m, 3H, OCH2Ph, H-OCH2Ph), 4.63 (t,1H, J ¼ 6.9 Hz, H-4), 4.80 (d,1H,
J ¼ 10.6 Hz, OCH2Ph), 5.03 (dd, 1H, J ¼ 2.1 Hz, J ¼ 6.6 Hz, H-5),
5.26e5.33 (m, 2H, 2 ꢂ H-200), 5.86 (ddd, 1H, J ¼ 7.5 Hz, J ¼ 10.1 Hz,
J ¼ 17.4 Hz, H-100), 6.74 (s, 1H, NH), 7.25e7.40 (m, 10H, Ph); 13C NMR
d
1.44 (s, 9H, 3 ꢂ CH3), 1.45 (s, 9H, 3 ꢂ CH3),
3.65e3.72 (m, 2H, 2 ꢂ H-30), 4.06 (dd, 1H, J ¼ 2.6 Hz, J ¼ 6.7 Hz, H-
10), 4.41e4.43 (m, 1H, H-5), 4.50 (d, 1H, J ¼ 12.1 Hz, OCH2Ph), 4.57
(d, 1H, J ¼ 12.1 Hz, OCH2Ph), 4.59 (d, 1H, J ¼ 10.7 Hz, OCH2Ph), 4.64
(d, 1H, J ¼ 10.7 Hz, OCH2Ph), 4.74 (dd, 1H, J ¼ 3.4 Hz, J ¼ 7.4 Hz, H-4),
4.94 (dt, 1H, J ¼ 4.1 Hz, J ¼ 6.7 Hz, H-20), 5.23 (d, 1H, J ¼ 10.3 Hz, H-
(100 MHz, CDCl3):
d
58.6 (C-4), 69.1 (C-20), 70.2 (C-30), 73.5

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D. Jackova et al. / Carbohydrate Research 451 (2017) 59e71
68
200), 5.29 (d, 1H, J ¼ 17.1 Hz, H-200), 5.78e5.86 (m, 1H, H-100),
4.1.16. (2R,3S,4S,5R,6E)-1,3-Bis(benzyloxy)-5-[(tert-
butoxycarbonyl)amino]-4-hydroxyoctadec-6-en-2-yl p-
methylbenzenesulfonate (5)
Tridec-1-ene (0.214 mL, 0.90 mmol) and the second generation
Grubbs catalyst (15.2 mg, 0.018 mmol) were added to a solution of
30 (0.11 g, 0.18 mmol) in dry CH2Cl2 (3.8 mL). After heating at reflux
for 2 h, the mixture was allowed to cool to room temperature, and
the solvent was evaporated. Chromatography of the residue on
silica gel (n-hexane/ethyl acetate, 7:1) gave 0.109 g (79%) of alkene 5
7.20e7.36 (m,10H, Ph); 13C NMR (100 MHz, CDCl3):
d
27.6 (3 ꢂ CH3),
27.8 (3 ꢂ CH3), 57.4 (C-4), 67.7 (C-30), 72.9 (C-20), 73.3 (OCH2Ph),
74.4 (OCH2Ph), 76.7 (C-10), 78.2 (C-5), 82.8 (Cq), 83.4 (Cq), 118.1 (C-
200), 127.7 (2 ꢂ CHPh), 127.8 (CHPh), 128.0 (CHPh), 128.2 (2 ꢂ CHPh),
128.4 (4 ꢂ CHPh), 134.8 (C-100), 136.9 (Ci), 137.4 (Ci), 148.8 (C¼O),
151.8 (C-2), 152.3 (C¼O). Anal. Calcd for C32H41NO9: C, 65.85; H,
7.08; N, 2.40. Found: C, 65.93; H, 6.99; N, 2.51.
as a colourless oil; [
1174, 1364, 1496, 1688, 1713, 2852, 2923, 3421 cmꢁ1
(400 MHz, CDCl3):
0.88 (t, 3H, J ¼ 6.8 Hz, CH3), 1.25e1.35 (m, 18H,
a
]
D [21] þ6.2 (c 0.29, CHCl3). IR (neat) ymax 1095,
;
1H NMR
4.1.14. tert-Butyl (4R,5S)-5-[(10S,20R)-10,30-bis(benzyloxy)-2'-
d
(tosyloxy)propyl]-2-oxo-4-vinyloxazolidine-3-carboxylate (28)
Alcohol 26 (0.131 g, 0.27 mmol) was dissolved in dry CH2Cl2
(0.6 mL) and TsCl (0.129 g, 0.675 mmol), Et3N (0.19 mL, 1.35 mmol)
and Me3N.HCl (13 mg, 0.135 mmol) were successively added at
room temperature. The mixture was stirred until completion of the
reaction (judged by TLC, 1 h), and was then diluted with CH2Cl2
(5 mL) before washing with a saturated NH4Cl solution (5 mL). The
organic layer was dried over Na2SO4, the solvent was evaporated in
vacuo, and the residue was flash-chromatographed on silica gel (n-
hexane/ethyl acetate, 5:1) to afford 0.164 g (95%) of compound 28
9 ꢂ CH2), 1.45 (s, 9H, 3 ꢂ CH3), 1.98e2.03 (m, 2H, CH2), 2.39 (s, 3H,
CH3), 2.90 (br s, 1H, OH), 3.56e3.60 (m, 1H, H-4), 3.69 (dd, 1H,
J ¼ 6.3 Hz, J ¼ 10.8 Hz, H-1), 3.79e3.84 (m, 2H, H-1, H-3), 4.28e4.40
(m, 3H, H-5, OCH2Ph), 4.53 (d, 1H, J ¼ 10.5 Hz, OCH2Ph), 4.71 (d, 1H,
J ¼ 10.5 Hz, OCH2Ph), 4.82 (d, 1H, J ¼ 8.5 Hz, NH), 5.12e5.14 (m, 1H,
H-2), 5.39 (dd, 1H, J ¼ 4.5 Hz, J ¼ 15.6 Hz, H-6), 5.57e5.65 (m, 1H, H-
7), 7.15e7.22 (m, 4H, Ph), 7.27e7.35 (m, 8H, Ph), 7.78e7.80 (m, 2H,
Ph); 13C NMR (100 MHz, CDCl3):
d 14.1 (CH3), 21.6 (CH3), 22.7 (CH2),
28.3 (3 ꢂ CH3), 29.2 (2 ꢂ CH2), 29.3 (CH2), 29.5 (CH2), 29.6
(2 ꢂ CH2), 29.7 (CH2), 31.9 (CH2), 32.3 (CH2), 53.2 (C-5), 67.9 (C-1),
72.7 (C-4), 73.2 (OCH2Ph), 74.4 (OCH2Ph), 79.2 (C-3), 79.6 (Cq), 81.9
(C-2), 127.3 (C-6), 127.6 (2 ꢂ CHPh), 127.7 (CHPh), 127.9 (CHPh), 128.1
(2 ꢂ CHPh), 128.3 (2 ꢂ CHPh), 128.4 (3 ꢂ CHPh), 128.7 (CHPh), 129.6
(2 ꢂ CHPh), 133.0 (C-7), 133.7 (Ci), 137.4 (Ci), 137.6 (Ci), 144.5 (Ci),
155.9 (C¼O). Anal. Calcd for C44H63NO8S: C, 68.99; H, 8.29; N, 1.83.
Found: C, 68.90; H, 8.36; N, 1.91.
as a colourless oil; [
1061, 1175, 1366, 1725, 1813, 2929, 2979 cmꢁ1; 1H NMR (400 MHz,
CDCl3):
a]
[21] þ12.7 (c 0.30, CHCl3). IR (neat) ymax
D
d
1.44 (s, 9H, 3 ꢂ CH3), 2.37 (s, 3H, CH3), 3.62 (dd, 1H,
J ¼ 5.0 Hz, J ¼ 10.9 Hz, H-30), 3.68 (dd, 1H, J ¼ 4.6 Hz, J ¼ 10.9 Hz, H-
30), 3.95 (t, 1H, J ¼ 4.2 Hz, H-10), 4.35e4.41 (m, 3H, H-5, OCH2Ph),
4.52 (d, 1H, J ¼ 11.0 Hz, OCH2Ph), 4.57 (d, 1H, J ¼ 11.0 Hz, OCH2Ph),
4.63 (dd, 1H, J ¼ 2.8 Hz, J ¼ 6.8 Hz, H-4), 4.82e4.85 (m, 1H, H-20),
5.23e5.27 (m, 2H, 2 ꢂ H-200), 5.81 (ddd, 1H, J ¼ 6.8 Hz, J ¼ 10.2 Hz,
J ¼ 17.1 Hz, H-100), 7.15e7.23 (m, 6H, Ph), 7.28e7.35 (m, 6H, Ph),
4.1.17. (2R,3S,4R,5S)-4-(Benzyloxy)-5-[(benzyloxy)methyl]-2-[(E)-
tridec-10-en-10-yl]pyrrolidin-3-ol (32)
7.73e7.75 (m, 2H, Ph); 13C NMR (100 MHz, CDCl3):
d
21.6 (CH3), 27.8
To an ice-cold solution of 5 (0.10 g, 0.13 mmol) in CH2Cl2
(0.40 mL) was added TFA (0.40 mL). After stirring at 0 ꢀC for 15 min,
the mixture was concentrated in vacuo, the obtained residue was
dissolved with MeOH (0.90 mL) and treated with aqueous NH3
solution (~26%) until basic pH (three times). Evaporation of the
solvents and chromatography of the crude product on silica gel (n-
hexane/ethyl acetate, 1:2) afforded 61 mg (96%) of compound 32 as
(3 ꢂ CH3), 57.8 (C-4), 67.6 (C-30), 73.4 (OCH2Ph), 74.2 (OCH2Ph), 77.3
(C-5), 77.4 (C-10), 78.2 (C-20), 83.6 (Cq),117.9 (C-200), 127.7 (2 ꢂ CHPh),
127.8 (2 ꢂ CHPh), 127.9 (CHPh), 128.1 (CHPh), 128.2 (2 ꢂ CHPh), 128.4
(4 ꢂ CHPh), 129.8 (2 ꢂ CHPh), 133.4 (Ci), 134.3 (C-100), 136.5 (Ci), 137.1
(Ci), 145.1 (Ci), 148.7 (C¼O), 151.5 (C-2). Anal. Calcd for C34H39NO9S:
C, 64.03; H, 6.16; N, 2.20. Found: C, 64.10; H, 6.09; N, 2.28.
a pale yellow oil; [
1027, 1095, 1454, 1670, 2851, 2921, 3404 cmꢁ1; 1H NMR (400 MHz,
CDCl3):
0.88 (t, 3H, J ¼ 6.8 Hz, CH3), 1.25e1.38 (m, 18H, 9 ꢂ CH2),
a
]
D [21] ꢁ11.9 (c 0.35, CHCl3). IR (neat) ymax 969,
d
4.1.15. (2R,3S,4S,5R)-1,3-Bis(benzyloxy)-5-[(tert-butoxycarbonyl)
amino]-4-hydroxyhept-6-en-2-yl p-methylbenzenesulfonate (30)
Cs2CO3 (81 mg, 0.25 mmol) was added to an ice-cold solution of
28 (0.153 g, 0.25 mmol) in EtOH (5.6 mL). After stirring for 2 h at
0 ꢀC, the mixture was diluted wit H2O (25 mL) and extracted with
EtOAc (3 ꢂ 30 mL). The combined organic layers were dried over
Na2SO4, the filtrate was concentrated in vacuo, and the residue was
purified by flash chromatography on silica gel (n-hexane/ethyl ac-
etate, 5:1) to provide 0.124 g (81%) of compound 30 as a colourless
2.01e2.06 (m, 2H, CH2), 3.10 (br s, 1H, OH), 3.39 (dd, 1H, J ¼ 3.4 Hz,
J ¼ 6.9 Hz, H-2), 3.48e3.52 (m, 1H, H-5), 3.57 (dd, 1H, J ¼ 3.9 Hz,
J ¼ 9.6 Hz, H-100), 3.64 (dd, 1H, J ¼ 3.8 Hz, J ¼ 9.6 Hz, H-100),
3.99e4.01 (m,1H, H-3), 4.12 (dd,1H, J ¼ 4.5 Hz, J ¼ 8.2 Hz, H-4), 4.50
(d, 1H, J ¼ 11.6 Hz, OCH2Ph), 4.53 (d, 1H, J ¼ 11.9 Hz, OCH2Ph), 4.58
(d, 1H, J ¼ 11.9 Hz, OCH2Ph), 4.71 (d, 1H, J ¼ 11.6 Hz, OCH2Ph),
5.58e5.72 (m, 2H, H-10, H-20), 7.24e7.37 (m, 10H, Ph); 13C NMR
(100 MHz, CDCl3):
d
14.1 (CH3), 22.7 (CH2), 29.2 (2 ꢂ CH2), 29.3
(CH2), 29.5 (CH2), 29.6 (2 ꢂ CH2), 29.7 (CH2), 31.9 (CH2), 32.6 (CH2),
58.3 (C-5), 62.7 (C-2), 68.3 (C-100), 71.8 (C-3), 72.6 (OCH2Ph), 73.6
(OCH2Ph), 80.5 (C-4),126.2 (C-10),127.7 (2 ꢂ CHPh),127.8 (4 ꢂ CHPh),
128.4 (4 ꢂ CHPh), 134.9 (C-20), 137.5 (Ci), 137.9 (Ci). Anal. Calcd for
oil; [
1496, 1689, 1713, 2850, 2920, 3420 cmꢁ1
CDCl3):
a
]
[21] þ3.1 (c 0.46, CHCl3). IR (neat) ymax 1095, 1174, 1364,
D
;
1H NMR (400 MHz,
d
1.45 (s, 9H, 3 ꢂ CH3), 2.39 (s, 3H, CH3), 2.95 (br s, 1H, OH),
3.63e3.70 (m, 2H, H-1, H-4), 3.80e3.84 (m, 2H, H-1, H-3),
4.34e4.40 (m, 3H, H-5, OCH2Ph), 4.53 (d, 1H, J ¼ 10.4 Hz, OCH2Ph),
4.71 (d, 1H, J ¼ 10.4 Hz, OCH2Ph), 4.87 (d, 1H, J ¼ 8.3 Hz, NH),
5.11e5.14 (m, 1H, H-2), 5.18e5.23 (m, 2H, 2 ꢂ H-7), 5.76e5.84 (m,
1H, H-6), 7.16e7.23 (m, 4H, Ph), 7.26e7.35 (m, 8H, Ph), 7.78e7.80
C32H47NO3: C, 77.85; H, 9.60; N, 2.84. Found: C, 77.94; H, 9.53; N,
2.90.
4.1.18. (2S,3R,4S,5R)-2-(Hydroxymethyl)-5-tridecylpyrrolidine-3,4-
diol hydrochloride (3)
(m, 2H, Ph); 13C NMR (100 MHz, CDCl3):
d
21.6 (CH3), 28.3 (3 ꢂ CH3),
To pyrrolidine derivative 32 (21 mg, 42.5
mixture of MeOH/EtOAc (3.5 mL, 3:1) was added 20% Pd(OH)2/C
(23.5 mg) followed by 12 M aqueous HCl solution (35 L) at room
mmol) dissolved in a
53.6 (C-5), 67.8 (C-1), 72.2 (C-4), 73.3 (OCH2Ph), 74.5 (OCH2Ph), 79.2
(C-3), 79.7 (Cq), 81.7 (C-2),116.1 (C-7),127.6 (2 ꢂ CHPh),127.8 (CHPh),
127.9 (CHPh), 128.0 (CHPh), 128.3 (2 ꢂ CHPh)) 128.4 (2 ꢂ CHPh), 128.7
(CHPh), 129.6 (2 ꢂ CHPh), 133.6 (2 ꢂ CHPh), 136.2 (C-6), 137.3 (Ci),
137.6 (2 ꢂ Ci), 144.6 (Ci), 155.9 (C¼O). Anal. Calcd for C33H41NO8S: C,
64.79; H, 6.76; N, 2.29. Found: C, 64.86; H, 6.68; N, 2.35.
m
temperature. After being stirred an atmosphere of hydrogen until
no starting material was observed (1 h, judged by TLC), the mixture
was filtered through a small pad of Celite. After the solvent was
removed, the residue was washed tree times with dry Et2O and

ꢀ
D. Jackova et al. / Carbohydrate Research 451 (2017) 59e71
69
dried under vacuum for 6 h to furnish compound 3 in the nearly
quantitative yield as a white amorphous solid; [
[22] ꢁ17.1 (c
0.28, MeOH). IR (neat) ymax 1031, 1085, 1144, 1462, 1572, 1638, 2849,
2919, 3335 cmꢁ1 1H NMR (600 MHz, CD3OD):
0.90 (t, 3H,
59.7 (C-4), 67.3 (C-30), 73.2 (OCH2Ph), 73.3 (OCH2Ph), 74.2 (C-5),
76.1 (C-10), 79.4 (C-20), 84.0 (Cq), 120.6 (C-200), 127.6 (2 ꢂ CHPh),127.8
(3 ꢂ CHPh), 128.0 (CHPh), 128.1 (2 ꢂ CHPh), 128.4 (4 ꢂ CHPh), 129.7
(2 ꢂ CHPh), 130.6 (C-100), 133.3 (Ci), 136.9 (Ci), 137.4 (Ci), 144.9 (Ci),
148.5 (C¼O), 150.9 (C-2). Anal. Calcd for C34H39NO9S: C, 64.03; H,
6.16; N, 2.20. Found: C, 64.11; H, 6.23; N, 2.14.
a]
D
;
d
J ¼ 7.0 Hz, CH3), 1.29e1.47 (m, 22H, 11 ꢂ CH2), 1.68e1.74 (m, 1H,
CH2), 1.87e1.93 (m, 1H, CH2), 3.37e3.40 (m, 1H, H-5), 3.63e3.67 (m,
1H, H-2), 3.84 (dd, 1H, J ¼ 8.9 Hz, J ¼ 12.0 Hz, H-100), 3.89 (dd, 1H,
J ¼ 4.3 Hz, J ¼ 12.0 Hz, H-100), 4.19e4.20 (m, 1H, H-4), 4.41 (dd, 1H,
4.1.21. (2R,3S,4S,5S)-1,3-Bis(benzyloxy)-5-[(tert-butoxycarbonyl)
amino]-4-hydroxyhept-6-en-2-yl p-methylbenzenesulfonate (31)
Using the same procedure as described for the preparation of 30,
compound 29 (96 mg, 0.15 mmol) and Cs2CO3 (49 mg, 0.15 mmol)
afforded after column chromatography on silica gel (n-hexane/
ethyl acetate, 4:1) 84 mg (92%) of derivative 31 as a colourless oil;
J ¼ 4.5 Hz, J ¼ 7.5 Hz, H-3); 13C NMR (150 MHz, CD3OD):
d 14.5
(CH3), 23.8 (CH2), 27.4 (CH2), 27.7 (CH2), 30.5 (2 ꢂ CH2), 30.6 (CH2),
30.7 (CH2), 30.8 (4 ꢂ CH2), 33.1 (CH2), 59.8 (C-100), 62.4 (C-5), 63.2
(C-2), 71.6 (C-3), 71.8 (C-4). Anal. Calcd for C18H38ClNO3: C, 61.43; H,
10.88; N, 3.98. Found: C, 61.35; H, 10.95; N, 3.92.
[
a
]
D [20] þ21.5 (c 0.53, CHCl3). IR (neat) ymax 1095, 1174, 1364, 1496,
4.1.19. tert-Butyl (4S,5S)-5-[(10R,20R)-10,30-bis(benzyloxy)-20-
hydroxypropyl]-2-oxo-4-vinyloxazolidine-3-carboxylate (27)
By the same procedure as described for the preparation of 26,
compound 8 (0.10 g, 0.26 mmol) was converted into derivative 27
1687, 1713, 2850, 2920, 3420 cmꢁ1 1H NMR (400 MHz, CDCl3):
;
d
1.42 (s, 9H, 3 ꢂ CH3), 2.36 (s, 3H, CH3), 3.67e3.71 (m, 3H, H-1, H-3,
H-4), 3.83 (dd, 1H, J ¼ 4.9 Hz, J ¼ 10.9 Hz, H-1), 4.27e4.30 (m, 1H, H-
5), 4.35 (d, 1H, J ¼ 11.8, OCH2Ph), 4.39 (d, 1H, J ¼ 11.8 Hz, OCH2Ph),
4.45 (d, 1H, J ¼ 11.2 Hz, OCH2Ph), 4.75 (d, 1H, J ¼ 11.2 Hz, OCH2Ph),
5.09e5.16 (m, 4H, H-2, 2 ꢂ H-7, NH), 5.66 (ddd, 1H, J ¼ 7.3 Hz,
J ¼ 9.9 Hz, J ¼ 16.8 Hz, H-6), 7.15e7.20 (m, 4H, Ph), 7.24e7.34 (m, 8H,
(0.112 g, 89%, n-hexane/ethyl acetate, 3:1, colourless oil); [
[22] þ8.0 (c 0.30, CHCl3). IR (neat) ymax 1062, 1156, 1315, 1726, 1805,
2923, 3486 cmꢁ1; 1H NMR (400 MHz, CDCl3):
a]
D
d
1.49 (s, 9H, 3 ꢂ CH3),
2.60 (d, 1H, J ¼ 3.6 Hz, OH), 3.61e3.62 (m, 2H, 2 ꢂ H-30), 3.88 (dd,
1H, J ¼ 4.4 Hz, J ¼ 7.5 Hz, H-10), 4.14e4.19 (m, 1H, H-20), 4.53 (m, 2H,
OCH2Ph), 4.57 (d, 1H, J ¼ 11.1 Hz, OCH2Ph), 4.66e4.72 (m, 2H, H-4,
H-OCH2Ph), 4.77 (t, 1H, J ¼ 7.5 Hz, H-5), 5.30 (d, 1H, J ¼ 17.1 Hz, H-
200), 5.35 (d, 1H, J ¼ 10.3 Hz, H-200), 5.87e5.95 (m, 1H, H-100),
Ph), 7.77e7.79 (m, 2H, Ph); 13C NMR (100 MHz, CDCl3):
d 21.5 (CH3),
28.3 (3 ꢂ CH3), 55.3 (C-5), 67.7 (C-1), 73.2 (OCH2Ph, C-4), 73.4
(OCH2Ph), 79.5 (C-3), 79.7 (Cq), 81.6 (C-2), 118.1 (C-7), 127.6
(2 ꢂ CHPh), 127.7 (CHPh), 127.8 (CHPh), 128.0 (3 ꢂ CHPh), 128.3
(5 ꢂ CHPh), 129.6 (2 ꢂ CHPh), 133.1 (C-6), 133.5 (Ci), 137.7 (Ci), 141.2
(Ci), 144.6 (Ci), 155.7 (C¼O). Anal. Calcd for C33H41NO8S: C, 64.79; H,
6.76; N, 2.29. Found: C, 64.69; H, 6.83; N, 2.35.
7.22e7.38 (m,10H, Ph); 13C NMR (100 MHz, CDCl3):
d
27.8 (3 ꢂ CH3),
60.2 (C-4), 70.1 (C-30), 70.2 (C-20), 73.4 (OCH2Ph), 73.6 (OCH2Ph),
75.3 (C-5), 76.8 (C-10), 83.7 (Cq), 119.9 (C-200), 127.7 (2 ꢂ CHPh), 127.8
(3 ꢂ CHPh), 127.9 (CHPh), 128.4 (4 ꢂ CHPh), 131.5 (C-100), 137.5
(2 ꢂ Ci), 148.6 (C¼O), 151.4 (C-2). Anal. Calcd for C27H33NO7: C,
67.06; H, 6.88; N, 2.90. Found: C, 66.96; H, 6.95; N, 2.96.
4.1.22. (2R,3S,4S,5S,6E)-1,3-Bis(benzyloxy)-5-[(tert-
butoxycarbonyl)amino]-4-hydroxyoctadec-6-en-2-yl p-
methylbenzenesulfonate (6)
During this reaction we also isolated 14 mg (9%) of tert-butyl
(4S,5S)-5-{(10S,20R)-10,30-bis(benzyloxy)-2'-[(tert-butoxycarbonyl)
oxy]propyl}-2-oxo-4-vinyloxazolidine-3-carboxylate as a colour-
By the same procedure as described for the preparation of 5
from 30, compound 31 (73 mg, 0.12 mmol) was converted after
stirring at reflux (2.5 h) to derivative 6 (73 mg, 79%, n-hexane/ethyl
less oil; [
1254, 1368, 1741, 1820, 2853, 2923 cmꢁ1
CDCl3):
a
]
[21] ꢁ7.6 (c 0.34, CHCl3). IR (neat) ymax 1058, 1155,
acetate, 5:1, colourless oil); [
ymax 1096, 1175, 1364, 1454, 1496, 1686, 2853, 2923, 3379 cmꢁ1; 1H
NMR (400 MHz, CDCl3):
0.88 (t, 3H, J ¼ 6.8 Hz, CH3), 1.26e1.33 (m,
a
]
D [21] ꢁ7.3 (c 0.40, CHCl3). IR (neat)
D
;
1H NMR (400 MHz,
d
1.48 (s,18H, 6 ꢂ CH3), 3.73 (dd,1H, J ¼ 5.2 Hz, J ¼ 9.3 Hz, H-
d
30), 3.77 (dd, 1H, J ¼ 4.3 Hz, J ¼ 9.3 Hz, H-30), 3.89 (dd, 1H, J ¼ 2.7 Hz,
J ¼ 8.4 Hz, H-10), 4.38 (d, 1H, J ¼ 10.5 Hz, OCH2Ph), 4.55 (m, 2H,
OCH2Ph), 4.68e4.79 (m, 3H, H-4, H-5, H-OCH2Ph), 5.31 (d, 1H,
J ¼ 17.2 Hz, H-200), 5.37e5.41 (m, 2H, H-20, H-200), 5.80e5.89 (m, 1H,
18H, 9 ꢂ CH2), 1.43 (s, 9H, 3 ꢂ CH3), 1.93e1.98 (m, 2H, CH2), 2.37 (s,
3H, CH3), 3.66e3.75 (m, 3H, H-1, H-3, H-4), 3.84 (dd, 1H, J ¼ 4.9 Hz,
J ¼ 10.9 Hz, H-1), 4.22e4.25 (m, 1H, H-5), 4.34 (d, 1H, J ¼ 11.7 Hz,
OCH2Ph), 4.39 (d, 1H, J ¼ 11.7 Hz, OCH2Ph), 4.45 (d, 1H, J ¼ 11.2 Hz,
OCH2Ph), 4.75 (d, 1H, J ¼ 11.2 Hz, OCH2Ph), 4.94 (d, 1H, J ¼ 8.1 Hz,
NH), 5.12e5.14 (m, 1H, H-2), 5.24e5.30 (m, 1H, H-6), 5.48e5.58 (m,
1H, H-7), 7.15e7.21 (m, 4H, Ph), 7.26e7.35 (m, 8H, Ph), 7.77e7.79
H-100), 7.25e7.38 (m, 10H, Ph); 13C NMR (100 MHz, CDCl3):
d 27.7
(3 ꢂ CH3), 27.9 (3 ꢂ CH3), 60.0 (C-4), 67.9 (C-30), 72.6 (OCH2Ph), 73.2
(OCH2Ph), 73.4 (C-20), 74.5 (C-5), 75.6 (C-10), 82.6 (Cq), 83.8 (Cq),
120.2 (C-200), 127.6 (3 ꢂ CHPh), 128.0 (CHPh), 128.1 (2 ꢂ CHPh), 128.4
(4 ꢂ CHPh),131.0 (C-100),137.1 (Ci),137.9 (Ci),148.7 (C¼O),151.1 (C-2),
152.8 (C¼O). Anal. Calcd for C32H41NO9: C, 65.85; H, 7.08; N, 2.40.
Found: C, 65.92; H, 7.15; N, 2.34.
(m, 2H, Ph); 13C NMR (100 MHz, CDCl3):
d 14.1 (CH3), 21.6 (CH3),
22.7 (CH2), 28.3 (3 ꢂ CH3), 29.1 (CH2), 29.2 (CH2), 29.3 (CH2), 29.5
(CH2), 29.6 (2 ꢂ CH2), 29.7 (CH2), 31.9 (CH2), 32.5 (CH2), 55.1 (C-5),
67.9 (C-1), 73.2 (OCH2Ph, C-4), 73.5 (OCH2Ph), 79.7 (C-3), 79.8 (Cq),
81.9 (C-2), 124.4 (C-6), 127.6 (3 ꢂ CHPh), 127.7 (CHPh), 127.8 (CHPh),
128.1 (2 ꢂ CHPh), 128.3 (2 ꢂ CHPh), 128.4 (3 ꢂ CHPh), 129.6
(2 ꢂ CHPh), 133.6 (Ci), 135.5 (C-7), 137.4 (Ci), 137.5 (Ci), 144.5 (Ci),
155.7 (C¼O). Anal. Calcd for C44H63NO8S: C, 68.99; H, 8.29; N, 1.83.
Found: C, 68.89; H, 8.37; N, 1.88.
4.1.20. tert-Butyl (4S,5S)-5-[(10S,20R)-10,30-bis(benzyloxy)-2'-
(tosyloxy)propyl]-2-oxo-4-vinyloxazolidine-3-carboxylate (29)
According to the same procedure described for the construction
of 28 from 26, compound 27 (0.102 g, 0.21 mmol) was transformed
to derivative 29 (0.116 g, 87%, n-hexane/ethyl acetate, 5:1, white
solid); mp 89e90 ꢀC; [
1107, 1203, 1317, 1352, 1728, 1797, 2924 cmꢁ1
CDCl3):
a
]
D [20] ꢁ10.5 (c 0.42, CHCl3). IR (neat) ymax
4.1.23. (2S,3S,4R,5S)-4-(Benzyloxy)-5-[(benzyloxy)methyl]-2-[(E)-
tridec-10-en-10-yl]pyrrolidin-3-ol (33)
According to the same procedure described for the preparation
of 32 from 5, compound 6 (61 mg, 0.08 mmol) was transformed to
derivative 33 (pale yellow solid, 34 mg, 86%, n-hexane/ethyl ace-
;
1H NMR (400 MHz,
d
1.48 (s, 9H, 3 ꢂ CH3), 2.39 (s, 3H, CH3), 3.65 (dd, 1H,
J ¼ 6.5 Hz, J ¼ 10.8 Hz, H-30), 3.72 (dd, 1H, J ¼ 5.5 Hz, J ¼ 10.8 Hz, H-
30), 3.94 (dd, 1H, J ¼ 2.1 Hz, J ¼ 9.2 Hz, H-10), 4.33e4.40 (m, 3H,
OCH2Ph, H-OCH2Ph), 4.56 (dd, 1H, J ¼ 7.2 Hz, J ¼ 9.2 Hz, H-5),
4.70e4.73 (m,1H, H-4), 4.82 (d, 1H, J ¼ 10.8 Hz, OCH2Ph), 5.02e5.06
(m, 1H, H-20), 5.26 (d, 1H, J ¼ 17.1 Hz, H-200), 5.36 (d, 1H, J ¼ 10.4 Hz,
H-200), 5.66e5.75 (m, 1H, H-100), 7.16e7.36 (m, 12H, Ph), 7.77e7.80
tate, 1:1); mp 46e47 ꢀC; [
a]
[21] ꢁ20.9 (c 0.21, CHCl3). IR (neat)
D
ymax 1093, 1122, 1204, 1454, 2849, 2920, 3025, 3327 cmꢁ1; 1H NMR
(400 MHz, CDCl3):
d
0.88 (t, 3H, J ¼ 6.9 Hz, CH3), 1.25e1.38 (m, 18H,
9 ꢂ CH2), 1.97e2.02 (m, 2H, CH2), 2.52 (br s, 1H, OH), 3.52e3.55 (m,
(m, 2H, Ph); 13C NMR (100 MHz, CDCl3):
d
21.7 (CH3), 27.9 (3 ꢂ CH3),
1H, H-2), 3.56e3.62 (m, 3H, H-5, 2 ꢂ H-100), 3.86 (t,1H, J ¼ 5.0 Hz, H-

ꢀ
D. Jackova et al. / Carbohydrate Research 451 (2017) 59e71
70
3), 4.06e4.09 (m, 1H, H-4), 4.52 (d, 1H, J ¼ 11.8 Hz, OCH2Ph), 4.56 (d,
1H, J ¼ 11.8 Hz, OCH2Ph), 4.60 (d, 1H, J ¼ 11.5 Hz, OCH2Ph), 4.66 (d,
1H, J ¼ 11.5 Hz, OCH2Ph), 5.35 (ddt, 1H, J ¼ 1.2 Hz, J ¼ 7.0 Hz,
J ¼ 15.2 Hz, H-10), 5.60e5.68 (m, 1H, H-20), 7.24e7.37 (m, 10H, Ph);
fibroblasts). A-549, HCT-116, MCF-7, MDA-MB-231, Caco-2, Jurkat
and HeLa cells were maintained in RPMI 1640 medium. NiH 3T3 cell
line was maintained in growth medium consisting of high glucose
Dulbecco's Modified Eagle Medium. Both of these media were
supplemented with Glutamax, and with 10% (V/V) foetal calf serum,
penicillin (100 IU ꢂ mLꢁ1), and streptomycin (100 mg ꢂ mLꢁ1) (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.
13C NMR (100 MHz, CDCl3):
d
14.1 (CH3), 22.7 (CH2), 29.2 (2 ꢂ CH2),
29.3 (CH2), 29.5 (CH2), 29.6 (3 ꢂ CH2), 31.9 (CH2), 32.3 (CH2), 58.0
(C-5), 65.2 (C-2), 69.4 (C-100), 73.5 (2 ꢂ OCH2Ph), 76.0 (C-3), 79.7 (C-
4), 127.7 (3 ꢂ CHPh), 127.8 (3 ꢂ CHPh), 128.4 (4 ꢂ CHPh), 129.7 (C-10),
132.8 (C-20), 137.8 (Ci), 138.0 (Ci). Anal. Calcd for C32H47NO3: C,
77.85; H, 9.60; N, 2.84. Found: C, 77.77; H, 9.66; N, 2.76.
4.2.2. Cytotoxicity assay
4.1.24. (2S,3R,4S,5S)-2-(Hydroxymethyl)-5-tridecylpyrrolidine-3,4-
diol hydrochloride (4)
The cytotoxic effect of the tested compounds was studied using
the colorimetric microculture assay with the MTT endpoint [19].
The amount of MTT reduced to formazan was proportional to the
number 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
Using the same procedure as described for the preparation of 4
from 32, compound 33 (25 mg, 50.6
rolidine (quantitative yield, white amorphous solid);
[20] ꢁ35.5 (c 0.22, MeOH). IR (neat) ymax 1051, 1129, 1466, 1638,
2850, 2920, 3390 cmꢁ1 1H NMR (400 MHz, CD3OD):
0.90 (t, 3H,
mmol) was converted to pyr-
4
[a]
D
10ꢁ4e10ꢁ6 mol ꢂ Lꢁ1. After 72 h incubation, 10
mL of MTT
;
d
(5 mg ꢂ mLꢁ1) were added into each well. After an additional 4 h,
J ¼ 6.7 Hz, CH3), 1.29e1.56 (m, 22H, 11 ꢂ CH2), 1.69e1.78 (m, 1H,
CH2),1.81e1.89 (m,1H, CH2), 3.43 (td,1H, J ¼ 5.4 Hz, J ¼ 9.2 Hz, H-5),
3.67e3.72 (m, 1H, H-2), 3.86e3.99 (m, 3H, H-4, 2 ꢂ H-100), 4.18 (t,
during which insoluble formazan was formed, 100 mL of 10% (m/m)
sodium dodecylsulfate were added into each well and another 12 h
were allowed for the formazan to be dissolved. The absorbance was
measured at 540 nm using the automated uQuant™ Universal
Microplate Spectrophotometer (Biotek Instruments Inc., Winooski,
VT USA). The blank corrected absorbance of the control wells was
taken as 100% and the results were expressed as a percentage of the
control.
1H, J ¼ 3.4 Hz, H-3); 13C NMR (100 MHz, CD3OD):
d 14.5 (CH3), 23.8
(CH2), 27.6 (CH2), 30.4 (CH2), 30.5 (2 ꢂ CH2), 30.7 (CH2), 30.8
(4 ꢂ CH2), 32.0 (CH2), 33.1 (CH2), 59.4 (C-100), 62.1 (C-5), 63.5 (C-2),
71.7 (C-3), 77.4 (C-4). Anal. Calcd for C18H38ClNO3: C, 61.43; H,
10.88; N, 3.98. Found: C, 61.52; H, 10.95; N, 3.92.
4.1.25. X-ray techniques
Single crystal of 24 suitable for X-ray diffraction was obtained
from a mixture of n-hexane/ethyl acetate by slow crystallization at
4
Acknowledgements
ꢀC. X-ray diffraction data were collected at 293 K on an Oxford
The present work was supported by the Scientific Grant Agency
(No. 1/0168/15 and No. 1/0871/16) of the Ministry of Education,
Slovak Republic. It was also supported by the Slovak Research and
Development Agency (SRDA Grant No. APVV-14-0883 and No.
APVV-15-0079), and by the project MediPark Kosice: 26220220185
supported by Operational Programme Research and Development
Diffraction Gemini R diffractometer equipped with a Ruby CCD
detector and Mo K sealed-tube source. Selected crystallographic
a
and other relevant data for compound 24 are listed in Table 1. Data
collection and reduction were performed with Oxford Diffraction
CrysAlis PRO version 171.38.43 software suite [20]. Crystal struc-
tures were solved by direct methods with charge-flipping algo-
ꢁ
(OPVaV-2012/2.2/08-RO, contract No. OPVaV/12/2013). We wish to
ꢁ
rithm Superflip [21] using
[22] and refined by least-squares
ꢀ
ꢀ
ꢀ
ꢁ
OLEX2
thank Dr. Maria Vilkova (P.J. Safarik University, Kosice) for NOESY
experiments.
procedure on F2 with SHELXL2013 [23]. All non-hydrogen atoms
were refined with anisotropic thermal parameters. Positions of all
H atoms were optimized under the constrain to ride on their parent
atoms, with CeH (aromatic) bond length of 0.93 Å, CeH (aliphatic)
bond length of 0.97 Å and bond length of 98 Å for hydrogen on
asymmetric carbon, OeH group bond length of 0.82 Å and NeH
group bond length of 0.86 Å. The DIAMOND programme was used for
the molecular graphics [24].
References
[1] For isolation and biological activity of broussonetines including diastereo-
meric broussonetinines, see: (a) D. Tsukamoto, M. Shibano, G. Kusano, Nat.
Med. 57 (2003) 68e72;
(b) D. Tsukamoto, M. Shibano, G. Kusano, Chem. Pharm. Bull. 49 (2001)
1487e1491;
(c) D. Tsukamoto, M. Shibano, R. Okamoto, G. Kusano, Chem. Pharm. Bull. 49
(2001) 492e496;
(d) M. Shibano, D. Tsukamoto, Y. Fujimoto, H. Masui, H. Sugimoto, G. Kusano,
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