Total synthesis of pachastrissamine together with its 4-epi-congener via [3,3]-sigmatropic rearrangements and antiproliferative/cytotoxic evaluation Dedicated to Associated Professor Ladislav Knie?o on the occasion of his 70th birthday

Article abstract of DOI:10.1016/j.carres.2014.11.004

Synthesis of the HCl salts of two anhydrophytosphingosines, jaspine B (1) and its 4-epi-congener 5 from easily available dimethyl l-tartrate and/or l-arabinose, is described. The key transformations are the efficient incorporation of a chiral amino group

Full text of DOI:10.1016/j.carres.2014.11.004

Accepted Manuscript
Total synthesis of pachastrissamine together with its 4-epi-congener via [3,3]-
sigmatropic rearrangements and antiproliferative/cytotoxic evaluation
Miroslava Martinková, Eva Mezeiová, Milica Fabiš íková, Jozef Gonda,
Martina Pilátová, Ján Mojžiš
PII:
S0008-6215(14)00420-0
DOI:
http://dx.doi.org/10.1016/j.carres.2014.11.004
CAR 6886
Reference:
To appear in:
Carbohydrate Research
Received Date:
Revised Date:
Accepted Date:
22 September 2014
10 November 2014
17 November 2014
Please cite this article as: Martinková, M., Mezeiová, E., Fabiš íková, M., Gonda, J., Pilátová, M., Mojžiš, J., Total
synthesis of pachastrissamine together with its 4-epi-congener via [3,3]-sigmatropic rearrangements and
antiproliferative/cytotoxic evaluation, Carbohydrate Research (2014), doi: http://dx.doi.org/10.1016/j.carres.
2
014.11.004
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Total synthesis of pachastrissamine together with its 4-epi-congener via
3,3]-sigmatropic rearrangements and antiproliferative/cytotoxic evaluation
[
a,∗
a
a
a
Miroslava Martinková , Eva Mezeiová , Milica Fabišíková , Jozef Gonda , Martina
b
b
Pilátová , Ján Mojžiš
a
Department of Organic Chemistry, Institute of Chemical Sciences, Faculty of Science, P.J. Šafárik
University, Moyzesova 11, 040 01 Košice, Slovak Republic
b
Department of Pharmacology, Faculty of Medicine, P.J. Šafárik University, SNP 1, 040 66 Košice,
Slovak Republic
th
Dedicated to Associated Professor Ladislav Kniežo on the occasion of his 70 birthday
ARTICLE INFO
Article history:
Received
Accepted
Available online
Keywords:
Anhydrophytosphingosines, Jaspine B, Pachastrissamine, [3,3]-Heterosigmatropic
rearrangements, Stereoselective synthesis, Anticancer activity
ABSTRACT
Synthesis of the HCl salts of two anhydrophytosphingosines, jaspine B (1) and its 4-epi-
congener 5 from easily available dimethyl L-tartrate and/or L-arabinose, is described. The key
transformations are the efficient incorporation of a chiral amino group via [3,3]-sigmatropic
rearrangements, a Wittig olefination for the instalment of the carbon backbone and the acid-
promoted building-up of
a
tetrahydrofuran framework. Evaluation for in vitro
antiproliferative/cytotoxic activity with a panel of human tumor cell lines using a MTT assay
revealed for some compounds of our strategy noteworthy activity. Compound 1.HCl (IC50: 0.
4
1-2.35 µM), its antipode ent-1.HCl (IC50: 4.07-5.69 µM) and also stereoisomer 4.HCl (IC50:
∗
Corresponding author. Tel.: +421-55-6222329: fax: +421-55-6222124.
E-mail address: miroslava.martinkova@upjs.sk (M. Martinková).

4
.28-6.10 µM) exhibited significant potency compared with clinically available anticancer
drugs such as cisplatin (IC50: 11.4-14.7 µM) and etoposide (IC50: 1.2-21.2 µM) on MDA-MB-
31, MCF-7 and Jurkat cells.
2
1. Introduction
Tetrahydrofuran ring systems are highly prevalent substructures found in many natural
1
bioactive molecules. Among these, a class of unusual sphingolipid tetrahydrofuran
derivatives substituted with an alkyl side chain functionality and possessing a vicinal amino
alcohol motif termed the anhydrophytosphingosines, have drawn much attention from the
synthetic and medicinal chemists owing to significant biological properties (vide infra). (+)-
Jaspine B (1) (also known as pachastrissamine, Fig. 1) is one of the anhydrophytosphingosine
2
analogues that has been isolated independently from two marine sponges (Pachastrissa sp.
3
3
and Jaspis sp. ) together with its oxazolidine congener, jaspine A (2, Fig. 1).
Figure 1. Structures of anhydrophytosphingosines.
2
,3
The structure of 1 was determined by extensive spectroscopic studies including NOE
2
experiments to be unprecedented sphingolipid related cyclic molecule, which is characterized
by the presence of a (2S,3S,4S)-configured tetrahydrofuran skeleton with the amino, hydroxyl
and C14 alkyl chain functionalities. All these structural features have been revealed to be
crucial for its biological activity (vide infra). Jaspine B has been reported to exhibit a
significant in vitro cytotoxicity against several different cancer cell lines including A-
2,3,4a-b
2
2
2,4c
4b,4d-e
4b
4f
4f
4g
5
49,
P-388, HT-29, MeL-28, MCF-7,
KB, HTC-116, U2OS, MDA-231,
4
g
4g
4e
4e
HeLa, CNE, MGC-803, EC-9706 with IC50 in the micromolar to sub-micromolar
ranges. It should be noted, that there are two reports incorporating the cytotoxicity of
4
b,4f
4b
4f
pachastrissamine 1,
its TFA salt and also antipode (ent-1) towards non-malignant cells,

4
b
4f
concretely NiH 3T3 (mouse fibroblasts) and GM-637 (human fibroblasts); to allow
4
f
4b
comparison, IC50 = 0.4 µM for 1 on GM-637 and IC50 = 7.80 µM for 1 on NiH 3T3.
4
c
Andrieu-Abadie’s studies have shown that 1 induces apoptosis on melanoma cells (murine
B16 and human MeL-28). This effect is due to its potent inhibitory activity against
sphingomyelin synthase (SMS), an essential enzyme involved in the biosynthesis of
5
sphingomyelins. With the aim to elucidate the structure-activity relationships in 1 and its
congeners, the role of the stereochemical arrangement of substituents on the tetrahydrofuran
4
a,4d,4f,6
4a
backbone on the biological profile has been investigated.
Delgado and co-workers
4
f
and also Génisson et al. confirmed that the absolute configuration of the tetrahydrofuran core
is the decisive factor for the cytotoxic properties. ent-Jaspine B (ent-1), for instance, was
4
f
4f
found to display a lower activity against some aforementioned cell lines [HTC-116, U2OS
4d
4a
and MCF-7 (1 in the form of TFA salt)]. Furthermore, compounds 3 (A-549 and MCF-
4d
4a
4a
7
), 4 (A-549 ) and ent-5 (A-549 ) were less potent than pachastrissamine, but still retained
the activity. To promote these aforementioned findings concerning the SAR, other studies
4
e,7
6b
focused on the side-chain modified analogues of 1
and 5, as well as the core ring-
8
modified derivatives of 1 have been developed. According to a recently Liu’s study, for
example, some 1,2,3-triazole-jaspine B hybrids were found to indicate excellent
4
e
6a
cytotoxicity. Further biological screening realized by Fujii et al. revealed that jaspine B
and all its isomers inhibit both forms of sphingosine kinase (SphK1 and SphK2) with
moderate to high activities, at which compounds ent-4 and 5 (Fig. 1) exhibited the most potent
inhibitory profile. The same authors also have demonstrated that SphK inhibitory activity
depends on the length of the hydrocarbon side chain and the naturally C14 carbon functionality
6
b
was found to be the most optimal.
The remarkable biological activity, simple, but unique structural features and limited
availability of the natural anhydrophytosphingosines have resulted in the development of
9
numerous total syntheses of 1, as well as its stereoisomers. In 2008, Davies et al. excellently
reviewed the isolation, characterization, stereochemical assignment, and syntheses of jaspine
B together with the construction of 2-epi-jaspine B (3). In recent six years a number of
synthetic chemists have undertaken studies on the preparation of pachastrissamine and its
analogues employing different approaches and various starting materials (Scheme 1).
However the most straightforward routes still appear to be those utilizing the chiral pool
1
0
11
commencing substrates, such as (S)-Garner’s aldehyde, (R)-Garner’s aldehyde, D-serine
1
2
13
14
15
16
methyl ester, D-glucose, D-xylose, D-ribose, 3-amino-3-deoxy-D-ribofuranose, D-
1
7
18
19
20
21
mannose, L-ascorbic acid, D-isoascorbic acid, D-erythronolactone, D-glyceraldehyde,

2
2
23
24
4a
4a
4a
L-tartaric acid, L-threitol, phytosphingosines (D-ribo-, L-lyxo-, L-arabino-, D-xylo- ),
2
5
26
(
R)-2-chlorohept-6-enal, (3R,4R)-hexa-1,5-diene-3,4-diol since majority of them include
parts of the chiral fragments that are present in the final products mentioned above. Further, a
few enantioselective syntheses starting from the simple molecules such as 2,2-dimethyl-1,3-
2
7
28
29
dioxan-5-one, (E)-4-(benzyloxy)but-2-en-1-ol and 2-vinyloxirane have been realized.
2
7
These procedures involved an organocatalytic aldol reaction, Sharpless asymmetric
2
8
29
epoxidation and an enantioselective palladium-catalyzed allylic amination as the key
transformations (Scheme 1).
9
Scheme 1. Published total syntheses of jaspine B together with its some isomers after Davies’s review.
Recently, we have reported the total synthesis of four pachastrissamine stereoisomers (ent-1,
1
4b
17
ent-5 and ent-3 and 4 as HCl salts) based on the concept to obtain biologically active and
complex natural products using [3,3]-heterosigmatropic rearrangements on allylic scaffolds
(
thiocyanates and trichloroacetimidates) derived from the monosaccharide templates, as the
key transformation for the effective incorporation of the chiral amino group. Until now, there
was only one report utilizing the aforementioned rearrangements for the construction of
2
2a
jaspine B published by Ichikawa et al. We present herein the preparation of two further
members of anhydrophytosphingosine family, jaspine B (1) and its unnatural 4-epi-analogue 5
1
4b
30
(
Fig. 1), using our successful approach starting from the synthon 16 (for the construction
3
1
of ent-16, see lit. ) which was elaborated from two different starting materials, L-arabinose
and dimethyl L-tartrate.

2. Results and discussion
2
2
.1. Chemistry
3
0
.1.1. Preparation of the chiral building block, acetonide 16
Because our developed strategy based on the application of the [3,3]-sigmatropic
rearrangements in natural product synthesis has employed the simple carbohydrates as the
30
chiral pool starting point, we began the synthesis of the required scaffold 16 (Scheme 2)
from inexpensive and readily available L-arabinose. Its regioselective tritylation (TrCl,
3
2
pyridine, DMAP) afforded 5-O-trityl-L-arabinofuranose 6 (54%) which was converted into
the isopropylidene derivative 7 in 74% yield by treatment with 2,2-dimethoxypropane (2,2-
DMP) and catalytic amounts of CSA in dry acetone. The configuration at the C-3 position in 7
3
3
was inverted through an oxidation‒stereoselective reduction sequence. Thus, IBX mediated
transformation of 6 resulted in the formation of the corresponding ulose 8 (85%) whose
4
reduction with NaBH in EtOH provided the requisite β-L-lyxofuranose 9 as a single
diastereoisomer in 93% yield (Scheme 2). The remaining hydroxyl group in 9 was protected
as the benzyl ether 10 (90%) using the conventional conditions (BnBr, NaH, DMF). Removal
of the O-trityl group in 10 (CSA, CH
2
Cl /MeOH) followed by benzoylation of 11 (74%)
2
produced derivative 12 in 97% yield. Cleavage of the 1,2-O-isopropylidene moiety in 12 was
achieved with 80% TFA to give lactol 13 (98%) whose sodium metaperiodate fragmentation
(
NaIO
).
4 2 4
, MeOH/H O) followed by NaBH treatment provided diol 14 in 84% yield (Scheme
2

Scheme 2. Reagents and conditions: (a) TrCl, DMAP, pyridine, 40 °C→rt; (b) 2,2-DMP, acetone, CSA, rt; (c)
IBX, MeCN, reflux; (d) NaBH
, EtOH, 0 °C→rt; (e) BnBr, DMF, NaH, TBAI, 0 °C→rt; (f) CSA,
CH Cl /MeOH (2:1), rt; (g) BzCl, DMAP, pyridine, 0 °C→rt; (h) 80% TFA, 0 °C; (j) (i) NaIO , MeOH/H
1:1), rt; (ii) NaBH , MeOH/CH Cl , 0° C→rt; (k) 2,2-DPM, CH Cl , p-TsOH, rt; (l) K CO , MeOH, 0 °C→rt.
4
2
2
4
2
O
(
4
2
2
2
2
2
3
Conversion of the generated 1,3-diol moiety in 14 to acetonide (2,2-dimethoxypropane, p-
TsOH, CH Cl ) led to the production of 15 (89%, Scheme 2) and the subsequent base-induced
hydrolysis of the ester functionality in 15 provided the desired derivative 16 in 95% yield
2
2
3
0
2
5
30
{
[α]
D
= +64.7 (c 0.30, CHCl
3
); lit. [α] = +64.0 (c 0.30, temperature and solvent not
D
reported)}. Because we required great amounts of the pure scaffold 16, the aforementioned
strategy did not appear to be practical; 12 reaction steps, recorded low, 14% overall yield.
Therefore, we looked for a more economic approach leading to 16 and adopted the known
3
0
three-step protocols, commencing from the commercially accessible dimethyl L-tartrate
3
0,34
(
Scheme 3). This sequence involved the production of the benzylidene derivative 17
84%), its reductive cleavage with LiAlH /AlCl system and the selective protection of the
(80%) which afforded 16 (24%) in 16% overall yield.
(
4
3
3
0,34
corresponding triol 18
4 3 2 2 2
Scheme 3. Reagents and conditions: (a) p-TsOH, PhCHO, benzene, reflux; (b) LiAlH , AlCl , Et O/CH Cl , 0
°
C→reflux; (c) 2-methoxypropene, PPTS, DMF, –15 °C→rt.
2
.1.2. [3,3]-Heterosigmatropic rearrangements
Having successfully completed the synthesis of 16, we turned our attention towards the
construction of suitable allylic substrates for the [3,3]-sigmatropic rearrangements. As shown
in Scheme 4, IBX oxidation of the hydroxyl group in 16 followed by a Horner-Wadsworth-
Emmons (HWE) olefination with triethyl phosphonoacetate (NaH, THF) provided an easily
separable mixture of α,β-unsaturated esters (E)-19 and (Z)-20 (E:Z = 88:12 ratio, as
1
determined by H NMR spectroscopy) in 81% and 12% isolated yields, respectively.
Geometry of the double bonds in these isomers was identified through the vinyl proton
coupling constant values; J3,2 = 15.7 Hz for (E)-19 and J3,2 = 11.8 Hz for (Z)-20. For further
transformations we used (E)-19 as the major product. Its reduction was achieved with DIBAl-
H in CH
2
Cl to furnish the corresponding alcohol 21 (93%, Scheme 4). On one hand,
2

mesylation of 21 (MsCl, Et
yield after two steps. On the other hand, alcohol 21 was transformed into trichloroacetimidate
3 using trichloroacetonitrile (CCl CN) and NaH in THF.
3
N) followed by KSCN treatment afforded thiocyanate 22 in 90%
2
3
Scheme 4. Reagents and conditions: (a) (i) IBX, MeCN, reflux; (ii) (EtO)
b) DIBAl-H, CH Cl N, CH Cl
, –50 °C→rt; (c) (i) MsCl, Et , 0 °C→rt; (ii) KSCN, MeCN, 0 °C→rt; (d) NaH,
Cl CCN, NaH, THF, 0 °C→rt; (e) Table 1; (f) Table 2.
2 2 2
P(O)CH CO Et, NaH, THF, 0 °C→rt;
(
2
2
3
2
2
3
As we had already prepared suitable substrates 22 and 23 from the common alcohol 21, our next task
was to realize individual rearrangements. The thermal reaction of thiocyanate 22 was carried
out in heptane at 70 °C and 90 °C and afforded the corresponding isothiocyanates 24 and 25
as a readily separable mixture of diastereoisomers in very good yields (85‒91%) and with
satisfactory selectivity (Table 1, entries 1-3).
Table 1
[
3,3]-Sigmatropic rearrangement of thiocyanate 22
a
b
c
Entry
Thiocyanate
Conditions
Time
h)
8
1.5
2
0.5
Ratio
Yield
(
24:25
90:10
86:14
85:15
76:24
(%)
85
91
91
88
1
2
3
4
22
22
22
22
∆, 70 °C
MW, 70 °C
∆, 90 °C
MW, 90 °C
a
In n-heptane.
b
c
1
Ratio in the crude reaction mixtures. Determined by H NMR spectroscopy.
Isolated combined yields.
On the other hand, the application of microwave energy at the same temperatures required
shorter reaction times (1.5 h and 0.5 h, entries 2 and 4, respectively, Table 1) and produced
the rearranged products 24 and 25 with yields and stereoselectivities similar to the thermal

driven reaction. Allylic imidate 23 rearranged using both the conventional thermal and
3
5
microwave irradiation conditions in o-xylene in the presence of K
2
CO
3
(Table 2). As
expected, two Owerman’s trichloroacetamides 26 and 27 were isolated from these reactions in
very good yields (75‒90%), but unfortunately none diastereoselectivity was observed (Table
2
, entries 1-4). As well as in the case of aza-Claisen reaction of 22, the use of microwave
heating was shown to be the most effective.
Table 2
Overman rearrangement of imidate 23
a
b
c
Entry
Imidate Conditions
Time
h)
3
1.5
0.5
52
Ratio
Yield
(
26:27
54:46
54:46
54:46
54:46
(%)
87
90
86
75
1
2
3
4
23
23
23
23
MW, 130 °C
MW, 150 °C
MW, 170 °C
∆, 140 °C
a
2 3
In o-xylene, in the presence of K CO .
Ratio in the crude reaction mixtures. Determined by H NMR spectroscopy.
Isolated combined yields.
b
c
1
2
.1.3. Synthesis of common oxazolidinones: confirmation of the stereochemistry
At this stage, the newly constructed stereochemistry in the key scaffolds 24-27 could be
1
4b
preliminarily assigned by analogy to previously obtained antipodes ent-24‒ent-27.
As
expected, the prepared rearranged products 24-27 had spectroscopic data in excellent
1
4b
agreement with those reported. The specific rotations were opposite in sign but matched
almost equal magnitudes (see experimental part) to those reported for the corresponding
1
4b
enantiomers. The configuration of the built chiral amino group in aza-Claisen product
series was later exactly determined by X-ray crystallographic analysis of the more advanced
derivative 28 and in the corresponding Overman product series by chemical correlations (vide
infra). Thus, for this purpose the major diastereoisomer 24 was through a two-step procedure
converted into crystalline derivative 28 in overall 83% yield (Scheme 5), the structure of
which has been unequivocally confirmed by a single crystal X-ray diffraction analysis (Fig. 2)
revealing (S) arrangement of the installed stereocentre. In a parallel fashion, the minor
isothiocyanate 25 was converted into carbamate 29 (89% yield over two steps, Scheme 5). To
achieve a synthesis of the common oxazolidinones 32 and 33, ozonolysis of both products 28
and 29 followed by a reductive work up (NaBH
4
) resulted in the formation of alcohols 30
(
81%) and 31 (87%) those NaH-mediated cyclization furnished the required cyclic derivatives
3
2 and 33 in 99% and 92% yields, respectively (Scheme 5).

Scheme 5. Reagents and conditions: (a) (i) MeONa, MeOH, 0 °C→rt, 97% from both 24 and 25; (ii) MNO,
MeCN, rt; (b) (i) O , MeOH or MeOH/CH Cl , –78 °C; (ii) NaBH , MeOH or MeOH/CH Cl , –78 °C→rt; (c)
NaH, THF, 0 °C→rt.
3
2
2
4
2
2
Figure 2. ORTEP structure of 28 showing the crystallographic numbering.
Having secured a reliable route to 32 and 33 from the products of aza-Claisen rearrangement
4 and 25, respectively, out next task was to build the same structures from the Overman’s
trichloroacetamides 26 and 27. It was achieved via a two-step approach involving the
ozonolysis followed by NaBH treatment to afford alcohols 34 (86%) and 35 (90%, Scheme
). Their elaboration with catalytic amounts of DBU induced the production of carbamates 32
2
4
6

and 33 in 95% and 96% yields, respectively. The prepared material had physical and
spectroscopic properties in excellent accord with those found for structures 32 and 33
previously obtained from isothiocyanates 24 and 25 confirming (S)-configured stereocentre
with nitrogen in the prevalent diastereoisomer 25.
O
O
OH
O
O
O
O
a
b
O
O
Cl C
NH OBn
Cl C
3
NH OBn
NH
OBn
3
26
34, 86%
32, 95%
O
O
O
O
OH
O
O
O
O
a
b
O
O
Cl C
3
NH OBn
Cl C
3
NH OBn
NH
OBn
O
27
O
35, 90%
33, 96%
3 2 2 4 2 2
Scheme 6. Reagents and conditions: (a) O , MeOH/CH Cl , –78 °C; (ii) NaBH , MeOH/CH Cl
–78 °C→rt; (b)
DBU, CH Cl
2
2
, 0 °C→rt.
Table 3
Crystal data and structure refinement parameters for compound 28
28
Empirical formula
18 5
C H25NO
Formula weight
Temperature, T (K)
Wavelength, λ (Å)
Crystal system
335.39
173(2)
0.71073
Orthorhombic
P 2
Space group
1 1 1
2 2
Unit cell dimensions
a (Å)
b (Å)
c (Å)3
V (Å )
8.7887(3)
9.0093(3)
22.5649(9)
1786.69(11)
4
Formula per unit cell, Z
3
Dcalcd (g/cm )
1.247
Absorption coefficient, µ
0.091
-
1
(mm )
F(0 0 0)
7
20
Crystal size (mm)
0.49 × 0.29 × 0.18
2.938-26.493
θ Range for data collection
o
(
)
Index ranges
–10 ≤ h ≤ 11
–
11 ≤ k ≤ 11

–
28 ≤ l ≤ 27
Independent reflections
3629(0.0280)
(Rint)
Absorption correction
Max. and min. transmission
Analytical
0
.987 and 0.970
Refinement method
Full-matrix least-squares
on F
2
Data/restraints/parameters
3
1
639/0/220
.055
2
Goodness-of-fit on F
Final R indices [I>2σ(I)]
R indices (all data)
R
R
1
= 0.0330, wR
= 0.0418, wR
2
= 0.0687
= 0.0724
1
2
Largest diff. peak and hole
0.136 and –0.178
-3
(e/Å )
2
.1.4. Coupling reaction and completion of the total synthesis
The modification of both oxazolidinone foldamers 32 and 33 to alcohols 42 and 43, as the
requisite handle for the attachment of the alkyl side chain, was a further task. As seen in
Scheme 7, the acid hydrolysis of the acetonide moiety in 32 and 33 in the presence of p-TsOH
in MeOH afforded the corresponding diols 36 (92%) and 37 (93%).Their treatment with trityl
chloride in dry pyridine resulted in the regioselective blocking of the primary hydroxyl group
to furnish derivatives 38 and 39 in 99% and 92% yields, respectively. The free remaining
functionalities in 38 and 39 were capped with a benzyl group to provide compounds 40 (94%)
and 41 (97%) before the subsequent transformations. Exposure of 40 and 41 to p-TsOH in
CH
2
Cl /MeOH resulted in the deprotection of the triphenylmethyl moiety delivering the
2
desired products 42 (93%) and 43 (97%, Scheme 7). IBX oxidation of the unprotected alcohol
function in 42 and 43 generated the corresponding aldehydes which immediately reacted with
3
6
destabilized Wittig reagent, derived from tridecyltriphenylphosphonium bromide and
LHMDS as a base, affording barely separable mixtures of olefins 44 and 45 (Z:E ≈ 88:12 for
1
4
4, Z:E ≈ 86:14 for 45, determined by H NMR spectroscopic analysis) with 83% and 91%
isolated yields, respectively (Scheme 7). Repeated chromatography of the small amounts of
the obtained mixtures of alkenes 44 and 45 furnished only (Z)-45 in pure form as analytical
sample. Having successfully constructed the carbon backbone with three neighbouring
stereogenic centres, all that remained was to build up a tetrahydrofuran skeleton. Thus, both
4
4
4 and 45 were hydrogenated under atmospheric pressure using 10% Pd/C as catalyst to give
6 (93%) and 47 (92%) with saturated alkyl chains and recovered secondary hydroxyl groups.
Subsequently, derivatives 46 and 47 were N-debenzylated by the catalytic hydrogenation at 60
C providing the corresponding protected L-lyxo- and L-xylo-phytosphingosines 48 (88%) and
°

4
9 (95%), respectively. Their exposure to 6 M HCl at reflux resulted in cleavage of the cyclic
carbamate fragment and concomitant cyclization afforded the HCl salts of 1 (89%) and 5
2
3
37
23
(
93%, Scheme 7). The [α]
D
value for 1.HCl {[α]
D
= +2.8 (c 0.36, MeOH); lit. [α]
D
=
1
4b
27
D
+
2.6 (c 0.38, MeOH); lit. [α]
= ‒2.9 (c 0.28, MeOH) for ent-1.HCl} and spectroscopic
data

Scheme 7. Reagents and conditions: (a) p-TsOH, MeOH, rt; (b) TrCl, DMAP, pyridine, 60 °C; (c) BnBr, NaH,
DMF, TBAI, 0 °C→rt; (d) p-TsOH, CH Cl /MeOH, rt; (e) (i) IBX, MeCN, reflux; (ii) LHMDS, C13 Br,
THF, rt; (f) H , 10% Pd/C, EtOH, rt; (g) H , 10% Pd/C, EtOH, 35% HCl, 60 °C; (h) 6 M HCl, reflux; (j) Ac O,
pyridine, DMAP, rt.
2
2
H27PPh
3
2
2
2
2
4
showed very good concordance with those previously reported. Compound 5.HCl {[α]
D
=
1
4b
27
D
‒
7.3 (c 0.46, MeOH); lit. [α]
magnitude for ent-5.HCl but was opposite in sign.
structure, we transformed 1.HCl to the known N,O-diacetate 50 (Ac
= +7.5 (c 0.52, MeOH)} also matched the published
1
4b
As an additional confirmation of
2
O, pyridine, DMAP,
2
2
18
26
8
1%). Again, its specific rotation {[α]
D
= ‒29.2 (c 0.24, CHCl
3
); lit. [α]
D
= ‒24.0 (c 0.60,
2
2a
23
37
23
38
22
CHCl
3
); lit. [α]
D
= ‒23.5 (c 0.40, CHCl
3
); lit. [α]
D
3 D
= ‒26.4 (c 0.50, CHCl ); lit. [α]
=
‒22.6 (c 1.00, CDCl
3
)} as well as spectroscopic data were in good agreement with those,
previously reported. In the same manner, compound 5.HCl was elaborated into fully
acetylated derivative 51 in 85% yield. The magnitude we obtained for specific rotation of 51
2
3
{
[α]
D
= ‒8.2 (c 0.15, CHCl )} was comparable with the values present in literature for ent-
3
1
4b
23
4d
28
5
1 {lit. [α]
D
= +9.9 (c 0.26, CHCl
3
); lit. [α]
D
3
= +7.4 (c 0.002, CHCl )} but had
opposite sense. In order to further verify the configuration of the prepared structures of
anhydrophytosphingosines 1.HCl and 5.HCl through their acetylated products 50 and 51,
respectively, NOE differential experiments were conducted. As shown in Fig. 3, in the case of
5
0: there is a series of larger enhancements between H-2 and H-3, between H-3 and H-4 and
between and H-2 and H-4 protons indicating that substituents on the tetrahydrofuran unit are
in the cis-relationship to each other. On the other hand, NOE experiments of 51 showed cis-
relationship between protons H-2 and H-3, and trans between H-3 and H-4 and also between
H-2 and H-4 protons on the tetrahydrofuran core (Fig. 3).
Figure 3. Some selected NOE enhancements for 50 and 51.

2
.2. Antiproliferative/cytotoxic activity
A
series of the prepared compounds (Table 4) was evaluated for the
antiproliferative/cytotoxic activities against six human cancer cell lines MDA-MB-231
(
(
mammary gland adenocarcinoma), MCF-7 (mammary gland adenocarcinoma), HTC-116
colon carcinoma), Caco-2 (colon carcinoma), Jurkat (acute T-lymphoblastic leukemia), and
HeLa (cervical adenocarcinoma); and a non-malignant cell line NiH 3T3 (mouse fibroblasts)
using the MTT assay. The obtained results are summarized in Table 4 as IC50 values.
Commercially available anticancer substances etoposide, cisplatin and doxorubicin were
included as positive control in the case of cell lines Jurkat, HeLa, MCF-7 and MDA-MB-
39
2
31 (Table 4), on HTC-116 and Caco-2 cell lines they were not tested. In an effort to find
new candidates with the enhanced anticancer activity and with the aim to compare, the
1
4b
aforementioned table also includes IC50 values for several derivatives (compounds: ent-24,
1
4b
14b
14b
14b
14b
14b
14b
14b
14b
ent-25, ent-32, ent-33, ent-46, ent-47, ent-48, ent-49, ent-1.HCl, ent-50,
1
4b
17
17
ent-51, ent-3.HCl and 4.HCl ) syntheses of which were reported previously.
Table 4
Antiproliferative activities on six human cancer cell lines (MDA-MB-231, MCF-7, HCT-116, Caco-2, Jurkat and
HeLa) and non-malignant mouse fibroblasts NiH 3T3
a
-1
Compd no.
Cell line, IC50 ± SD (µmol × L )
HCT-116 Caco-2 Jurkat
39.66± 3.88 14.00±4.47 ND
MDA-MB-231 MCF-7
HeLa
NiH 3T3
b
ent-24
40.26±5.59 25.40±4.70 31.63±0.99
38.49±7.94 23.26±4.55 23.77±3.74
61.04±8.74
b
ent-25
29.59±4.88
>100
6.25±1.37
>100
34.88±3.30 40.85±8.57
b
ent-32
>100
>100
>100
>100
>100
>100
>100
b
ent-33
>100
>100
>100
>100
>100
b
ent-46
8.61±1.24
>100
11.80±4.96 15.81±1.92
>100 >100
16.80±10.34 7.06±0.19
9.07±0.55
>100
7.76±0.88
>100
b
ent-47
>100
>100
b
ent-48
37.35±5.59 32.85±0.44 37.34±6.88
38.34±4.61 31.63±2.20 30.77±0.96
40.07±4.55
27.68±1.26
5.77±3.19
>100
28.93±2.66 46.20±12.23 35.06±6.87
26.14±2.37 ND 33.88±3.71
16.08±6.68 8.05±1.16
b
ent-49
b
ent-1.HCl
5.69±1.74
>100
4.07±2.54
>100
5.83±0.41
>100
5.66±2.14
>100
b
ent-50
>100
>100
b
ent-51
28.64±0.29 28.04±0.62 27.77±0.54
68.04±9.69 46.07±2.15 46.09±2.86
45.96±5.59 23.99±6.58 25.11±3.66
30.20±0.14
64.62±4.44
29.65±3.66
>100
28.03±1.07 >100
34.79±10.08
2
2
3
4
4
4
4
5
2
6
7
8
24.77±6.91 34.90±4.05 75.27±3.73
7.54±2.11
>100
32.41±7.87 42.21±15.15
>100 >100
25.00±9.08 14.85±5.33
>100
17.83±8.34 ND
>100 >100
>100
>100
17.93±2.73
>100
11.40±3.51
>100
7.46±0.30
>100
>100
>100
41.13±8.77 33.15±1.57 40.09±1.71
43.26±10.17 29.12±1.98 >100
33.57±8.77

4
1
9
44.45±4.19 38.07±1.58 31.14±1.40
34.36±3.46
0.35±0.11
(13.14)
>100
31.12±4.26 32.32±4.44 37.44±2.37
.HCl
2.35±1.20
>100
0.41±0.09
2.59±1.02
>100
0.50±0.27
(9.20)
>100
0.61±0.27
(7.54)
>100
4.60±0.90
>100
e
e
e
e
(11.22)
5
5
4
0
>100
1
34.06±3.92 29.33±0.78 27.52±2.08
6.10±1.52 4.30±2.08 6.41±1.70
21.94±1.90 12.50±5.06 8.44±1.09
32.65±0.81
3.73±2.21
5.96±0.51
NT
26.86±1.20 37.18±4.39 37.06±5.71
c
.HCl
4.28±1.39
6.45±1.06
12 ±1.8
19.24±9.32 8.02±0.14
23.28±1.31 17.30±6.60
c
ent-3.HCl
d
cisplatin
14.7±2.7
21.2±4.2
11.4±2.4
10.9±2.1
0.5±0.024
NT
NT
NT
7.7±2.3
3.9±2.3
NT
NT
NT
d
etoposide
NT
1.2±1.5
d
doxorubicin 0.2±0.8
NT
0.078±0.02 0.2±0.06
NT-not tested.
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.
Prepared according the reported procedures.
Prepared according the reported procedures.
b
c
d
14b
17
Values for the clinically available anticancer drugs are reported in ref. 39 and were utilized from the same
source.
e
Selectivity index (SI = IC50 against NiH 3T3 cells/IC50 against cancer cells).
To the best of our knowledge, this is the first report implementing the
antiproliferative/cytotoxic activity of jaspine B in the form of HCl salt (1.HCl) and also its
stereoisomeric analogues (ent-1.HCl, 4.HCl, and ent-3.HCl) towards human cancer cell line
Caco-2. Compound 1.HCl exhibited remarkable in vitro potency against all tested cancer cell
lines (IC50 values between 0.35 and 2.59 µM), especially on Caco-2 (colon carcinoma) in
micro- and sub-micromolar ranges and proved to be significantly more potent than its
enantiomer ent-1.HCl (IC50 values between 4.07 and 16.08 µM). Similarly, substances 4.HCl
and ent-3.HCl (stereoisomers of 1) have exhibited comparable or higher (in the case of
4
1
.HCl) in vitro activity than ent-1.HCl (Table 4). Moreover, compounds 4.HCl and ent-
.HCl displayed greater the antiproliferative/cytotoxic activity against MCF-7 and MDA-
MB-231 breast cancer cell lines than commercially available anticancer agents cisplatin and
etoposide. Notably, IC50 value (0.41 µM) for the natural compound 1.HCl on MCF-7 cells
39
was comparable to that published for doxorubicin (IC50 = 0.5 µM). These aforementioned
results reveal that the stereochemical arrangement of the tetrahydrofuran skeleton is one of
determining factors for the antiproliferative/cytotoxic activity and the study of the unnatural
derivatives (compounds ent-1.HCl, 4.HCl and ent-3.HCl) may be useful for further structural
optimization with a view to establish new candidates with enhanced anticancer activity. In
Table 4 are also incorporated IC50 values for the precursors, from which jaspine B and its
1
4b
antipode ent-1.HCl were constructed. Of them, only compound ent-46, which represents

the protected form of D-lyxo-phytospinhgosine exhibited good activity, mainly against MCF-
, MDA-MB-231 and Jurkat cancer cell lines with IC50 values comparable to those reported
7
3
9
for cisplatin, while its enantiomer, compound 46 was found to be less active (Table 4).
1
4b
Worthy of note, compounds 25 and ent-25 were at least 1.5× more potent than cisplatin on
Jurkat cells.
The antiproliferative/cytotoxic activity of substances included in Table 4 was also screened on
non-malignant mouse fibroblasts NiH 3T3. Among them, compounds 1.HCl, 4.HCl, ent-
1
.HCl and ent-3.HCl demonstrated in some cases a quite promising selectivity of the
aforementioned effects against cancer cells relative to the non-malignant NiH 3T3 cells. As
seen in Table 4, the high selectivity index (SI), calculated as IC50 for NiH 3T3 divided by IC50
for the corresponding cancer cells, was found on MCF-7 (SI = 11.22), Caco-2 (SI = 13.14),
Jurkat (SI = 9.20) and HeLa (SI = 7.54) cell lines for compound 1.HCl.
3
. Conclusions
In summary, we have developed a straightforward route to two anhydrophytosphingosines,
jaspine B (1) and its 4-epi-analogue 5, from easily available dimethyl L-tartrate and/or L-
arabinose. The described synthesis relies on two key reactions which are [3,3]-sigmatropic
rearrangements affording the required chiral amino group and the acid-promoted
intramolecular cyclization providing the tetrahydrofuran ring. Moreover, the elaborated
strategy allowed for the creation of two phytosphingosine molecules 48 and 49 with L-lyxo
and L-xylo configurations, respectively. Further, used Wittig olefination permits variations in
the length of the side chain to synthesize diverse analogues. A series of the prepared
derivatives was assessed for the antiproliferative/cytotoxic activity against six cancer cell
lines (Jurkat, HeLa, MDA-MB-231, MCF-7, HTC-116 and Caco-2) using the MTT assay.
Among them, compounds ent-46, ent-1.HCl, 1.HCl, 4.HCl and ent-3.HCl were the most
promising molecules and were selected as candidates for further screening and evaluation. In
terms of the selectivity index (SI), structures, which were found to be the most potent, will be
also examined on human non-malignant endothelial cells HUVEC (human umbilical vein
endothelial cells) and progress will be reported in due course.
4
. Experimental

4
.1. Chemistry
All commercial reagents were used in the highest available purity from Aldrich, Fluka, Merck
or Acros Organics without further purification. Solvents were dried and purified before use
according to standard procedures. For flash column chromatography on silica gel, Kieselgel
6
0 (0.040-0.063 mm, 230-400 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
2 4
acid, a basic potassium permanganate solution, or a solution of concentrated H SO , with
1
13
subsequent heating. H NMR and C NMR spectra were recorded in CDCl
3
, CD
3
OD and
1
13
C
6
D
6
on a Varian Mercury Plus 400 FT NMR (400.13 MHz for H and 100.61 MHz for C)
1
13
or on a Varian Premium COMPACT 600 (599.87 MHz for H and 150.84 MHz for C)
1
spectrometer using TMS as internal reference. For H, δ are given in parts per million (ppm)
relative to TMS (δ = 0.0), CD
3
OD (δ = 4.84), C
(δ = 77.00), CD OD (δ = 49.05), C
δ = 30.83). The multiplicity of the C NMR signals concerning the C- H coupling was
6
D
6
(δ = 7.15) and CD
3
COCD
3
(δ = 2.05) and
1
3
for C relative to CDCl
3
3
6
D
6
(δ = 128.02) and CD
3
COCD
3
1
3
13
1
(
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
-1
spectrometer and expressed in v values (cm ). Optical rotations were measured on a P-2000
Jasco polarimeter and reported as follows: [α] (c in grams per 100 mL, solvent). Melting
D
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 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 compressed
air. Small quantities of reagents (µL) were measured with appropriate syringes (Hamilton).
All reactions were performed under an atmosphere of nitrogen, unless otherwise noted.
4
.1.1. 1,2-O-Isopropylidene-5-O-trityl-β-L-arabinofuranose (7)
To a suspension of L-arabinose (2.50 g, 16.65 mmol) in dry pyridine (20 mL) were
successively added trityl chloride (4.64 g, 16.65 mmol) and DMAP (1.02 g, 8.33 mmol). The
resulting mixture was stirred 72 h at room temperature, then concentrated and co-evaporated
2 2
three times with toluene. The residue obtained was partitioned between CH Cl (40 mL) and a

saturated NaHCO
CH Cl (2 × 40 mL). The combined organic layers were dried over Na
taken down, and the residue was subjected to flash chromatography through a short column of
3
solution (25 mL), the aqueous phase was extracted with further portions of
2
2
2
SO , the solvent was
4
3
2
silica gel (n-hexane/ethyl acetate, 1:3) to afford 3.53 g (54%) of lactol 6 as a colourless
3
2
foam. The material had spectroscopic data in accord with those reported previously.
To a solution of 6 (3.51 g, 8.94 mmol) in dry acetone (23 mL) were successively added 2,2-
dimethoxypropane (23 mL) and a catalytic amount of CSA (16.6 mg, 0.07 mmol). After
stirring for 3 h at room temperature, MeOH (0.35 mL) was added, and the mixture was
partitioned between CHCl
layer was dried over Na
chromatographed on silica gel (n-hexane/ethyl acetate, 3:1) to furnish 2.86 g (74%) of
3
(105 mL) and a saturated NaHCO
3
solution (32 mL). The organic
2
SO , the solvent was evaporated in vacuo, and the residue was
4
2
5
40
21
compound 7 as a white foam; {[α]
ent-7}. IR (neat) υmax 3415, 3057, 2936, 1596, 1448, 1374, 1210, 1065 cm ; H NMR (400
MHz, CDCl ): δ 1.16 (s, 3H, CH ), 1.24 (s, 3H, CH ), 2.13 (br s, 1H, OH), 3.25 (dd, 1H, J5,5
.3 Hz, J5,4 = 7.7 Hz, H-5), 3.40 (dd, 1H, J5,5 = 9.3 Hz, J5,4 = 5.8 Hz, H-5), 4.15 (ddd, 1H, J5,4
D
+5.5 (c 0.40, CHCl
3
); lit [α]
D
3
‒1.0 (c 1.0, CHCl ) for
-1 1
3
3
3
=
9
=
7.7 Hz, J5,4 = 5.8 Hz, J4,3 = 2.3 Hz, H-4), 4.32 (m, 1H, H-3), 4.49 (d, 1H, J2,1 = 4.0 Hz, H-
1
3
2
2
1
), 5.87 (d, 1H, J2,1 = 4.0 Hz, H-1), 7.20–7.45 (m, 15H, Ph); C NMR (100 MHz, CDCl
6.0 (CH ), 26.6 (CH ), 63.8 (C-5), 76.6 (C-3), 86.6 (C-4), 2 × 86.8 (C-2, C ), 105.6 (C-1),
12.4 (C ), 127.0 (3 × CHPh), 127.8 (6 × CHPh), 128.6, (6 × CHPh), 143.7 (3 × C ). Anal. Calcd
: C, 74.98; H, 6.53. Found: C, 75.05; H, 6.48.
3
): δ
3
3
q
q
i
for C27
H
28
O
5
4
.1.2. 1,2-O-Isopropylidene-5-O-trityl-β-L-erythro-pentofuranos-3-ulose (8)
o-Iodoxybenzoic acid (3.64 g, 13.01 mmol) was added to a solution of 7 (2.80 g, 6.50 mmol)
in acetonitrile (60 mL), and the resulting suspension was stirred at reflux for 30 min. The
mixture was allowed to cool to room temperature, the insoluble material was filtered off, and
the filtrate was concentrated. The residue was purified by flash chromatography on silica gel
2
2
(
n-hexane/ethyl acetate, 5:1) to give 2.37 g (85%) of ulose 8 as a colourless foam; [α]
D
-
1
+
11.4 (c 0.28, CHCl
3
). IR (neat) υmax 3422, 2937, 1771, 1597, 1447, 1375, 1216, 1076 cm ;
): δ 1.32 (s, 3H, CH ), 1.36 (s, 3H, CH ), 3.37 (dd, 1H, J5,5 = 10.3
1
H NMR (400 MHz, CDCl
3
3
3
Hz, J5,4 = 4.0 Hz, H-5), 3.45 (dd, 1H, J5,5 = 10.3 Hz, J5,4 = 7.3 Hz, H-5), 4.32 (dd, 1H, J5,4
=
=
7
4
.3 Hz, J5,4 = 3.8 Hz, H-4), 4.36 (dd, 1H, J2,1 = 4.2 Hz, J4,2 = 0.5 Hz, H-2), 6.03 (d, 1H, J2,1
1
3
.2 Hz, H-1), 7.20–7.48 (m, 15H, Ph); C NMR (100 MHz, CDCl
3
): δ 26.7 (CH
3
), 27.3
(
CH
3
), 64.4 (C-5), 76.6 (C-2), 80.9 (C-4), 87.0 (C ), 102.7 (C-1), 115.0 (C ), 127.0 (3 ×
q
q

CHPh), 127.8 (6 × CHPh), 128.7 (6 × CHPh), 143.4 (3 × C
i
), 207.0 (C=O). Anal. Calcd for
27 26 5
C H O
: C, 75.33; H, 6.09. Found: C, 75.39; H, 6.04.
4
.1.3. 1,2-O-Isopropylidene-5-O-trityl-β-L-lyxofuranose (9)
To a solution of 8 (2.26 g, 5.25 mmol) in EtOH (75 mL) that had been pre-cooled to 0 °C was
added NaBH (0.397 g, 10.50 mmol). The resulting mixture was stirred at 0 °C for 10 min and
then for another 30 min at room temperature before evaporation of the solvent. The residue
obtained was partitioned between a saturated NaCl (68 mL) solution and CH Cl (95 mL), the
aqueous layer was washed with another portion of CH Cl (95 mL). The combined organic
extracts were dried over Na SO , the solvent was removed under reduced pressure, and the
4
2
2
2
2
2
4
crude product was subjected to flash chromatography through a short column of silica gel (n-
hexane/ethyl acetate, 4:1) to afford 2.12 g (93%) of compound 9 as white crystals; mp 61–63
2
4
°
C (recrystallized from n-hexane/ethyl acetate); [α]
D
+11.1 (c 0.66, CHCl
497, 3057, 2935, 1448, 1373, 1209, 1073 cm ; H NMR (400 MHz, CDCl
), 1.32 (s, 3H, CH ), 2.73 (d, 1H, J3,OH = 7.2 Hz, OH), 3.44 (dd, 1H, J5,5 = 9.8 Hz, J5,4
.5 Hz, H-5), 3.49 (dd, 1H, J5,5 = 9.8 Hz, J5,4 = 6.4 Hz, H-5), 4.22–4,31 (m, 2H, H-3, H-4),
.56 (dd, 1H, J3,2 = 5.6 Hz, J2,1 = 4.1 Hz, H-2), 5.72 (d, 1H, J2,1 = 4.1 Hz, H-1), 7.19–7.49 (m,
3
). IR (neat) υmax
-1 1
3
3
): δ 1.27 (s, 3H,
CH
3
3
=
5
4
1
7
1
7
13
5H, Ph); C NMR (100 MHz, CDCl
9.6 (C-2), 80.5 (C-4), 87.0 (C ), 104.8 (C-1), 114.0 (C
28.7 (6 × CHPh), 143.8 (3 × C ). Anal. Calcd for C27
4.93; H, 6.60.
3
): δ 26.5 (CH
3
), 26.6 (CH
), 127.0 (3 × CHPh), 127.8 (6 × CHPh),
: C, 74.98; H, 6.53. Found: C,
3
), 63.0 (C-5), 70.9 (C-3),
q
q
i
28 5
H O
4
.1.4. 3-O-Benzyl-1,2-O-isopropylidene-5-O-trityl-β-L-lyxofuranose (10)
To a solution of 9 (2.06 g, 4.76 mmol) in dry DMF (11.50 mL) that had been pre-cooled to 0
C were successively added NaH (0.171 g, 7.14 mmol, 60% dispersion in mineral oil), BnBr
0.68 mL, 5.72 mmol) and TBAI (45.20 mg, 0.01 mmol). The resulting mixture was stirred at
°C for 10 min, and then for a further 20 min at room temperature. The excess hydride was
decomposed by the addition of MeOH (0.5 mL), the mixture was poured into ice-water (20
mL) and extracted with Et O (2 × 30 mL). The combined organic layers were dried over
Na SO , the solvent was evaporated in vacuo, and the residue was chromatographed on silica
gel (n-hexane/ethyl acetate, 11:1) to give 2.24 g (90%) of compound 10 as a white foam;
°
(
0
2
2
4
2
2
-1
[
α]
D
–13.5 (c 0.40, CHCl
3
). IR (neat) υmax 3058, 2933, 1596, 1448, 1373, 1208, 1092 cm ;
1
H NMR (400 MHz, CDCl
3
): δ 1.18 (s, 3H, CH ), 1.24 (s, 3H, CH ), 3.45 (dd, 1H, J5,5 = 10.4
3
3
Hz, J5,4 = 3.5 Hz, H-5), 3.62 (dd, 1H, J5,5 = 10.4 Hz, H5, J5,4 = 7.5 Hz, H-5), 4.01 (dd, 1H, J4,3

=
7.3 Hz, J3,2 = 5.1 Hz, H-3), 4.38 (dt, 1H, J5,4 = 7.4 Hz, J4,3 = 7.4 Hz, J5,4 = 3.5 Hz, H-4),
.47 (d, 1H, JH,H = 12.1 Hz, OCH Ph), 4.53 (dd, 1H, J3,2 = 5.0 Hz, J2,1 = 4.0 Hz, H-2), 4.54 (d,
H, JH,H = 12.1 Hz, OCH Ph), 5.71 (d, 1H, J2,1 = 4.0 Hz, H-1), 7.13–7.52 (m, 20H, Ph); C
): δ 25.9 (CH ), 26.2 (CH ), 63.8 (C-5), 72.4 (OCH Ph), 77.2 (C-3),
8.4 (C-2), 80.4 (C-4), 86.7 (C ), 104.6 (C-1), 113.4 (C ), 126.8 (3 × CHPh), 127.7 (8 ×
CHPh), 127.8 (CHPh), 128.3 (CHPh), 128.8 (7 × CHPh), 137.4 (C ), 144.2 (3 × C ). Anal. Calcd
for C34 : C, 78.14; H, 6.56. Found: C, 78.10; H, 6.60.
4
1
2
13
2
NMR (100 MHz, CDCl
3
3
3
2
7
q
q
i
i
34 5
H O
4
.1.5. 3-O-Benzyl-1,2-O-isopropylidene-β-L-lyxofuranose (11)
Camphorsulfonic acid (46 mg, 0.20 mmol) was added to a solution of 10 (2.07 g, 3.96 mmol)
in a mixture of CH Cl /MeOH (2:1, 16 mL), and the resulting solution was stirred at room
temperature for 2.5 h. The reaction was quenched by neutralization with Na CO , the solvents
were evaporated, and the residue was chromatographed on silica gel (n-hexane/ethyl acetate,
2
2
2
3
2
:1) to yield 0.82 g (74%) of compound 11 as white crystals mp 56.5–58 °C (recrystallized
2
4
from n-hexane/ethyl acetate); [α]
D
+53.8 (c 0.32, CHCl
3
). IR (neat) υmax 3510, 2993, 2956,
): δ 1.34 (s, 3H, CH ),
), 2.32 (br s, 1H, OH), 3.91‒3.96 (m, 2H, 2 × H-5), 4.18 (dd, 1H, J4,3 = 7.5
Ph), 4.62
Ph), 5.75 (d, 1H,
): δ 25.9 (CH ), 26.6
Ph), 77.8 (C-3), 77.9 (C-2), 80.4 (C-4), 104.9 (C-1), 113.7
20 5
), 127.8 (2 × CHPh), 128.1 (CHPh), 128.6 (2 × CHPh), 137.1 (C ). Anal. Calcd for C15H O :
-
1 1
2
1
941, 2889, 1454, 1373, 1208, 1145 cm ; H NMR (400 MHz, CDCl
3
3
.63 (s, 3H, CH
3
Hz, J3,2 = 5.1 Hz, H-3), 4.22–4.27 (m, 1H, H-4), 4.57 (d, 1H, JH,H = 11.9 Hz, OCH
2
(
dd, 1H, J3,2 = 5.0 Hz, J2,1 = 4.1 Hz, H-2), 4.78 (d, 1H, JH,H = 11.9 Hz, OCH
2
13
J
2,1 = 4.0 Hz, H-1), 7.26–7.39 (m, 5H, Ph); C NMR (100 MHz, CDCl
3
3
(
(
CH
3
), 62.3 (C-5), 72.6 (OCH
2
C
q
i
C, 64.27; H, 7.19. Found: C, 64.32; H, 7.15.
4
.1.6. 5-O-Benzoyl-3-O-benzyl-1,2-O-isopropylidene-β-L-lyxofuranose (12)
To a solution of 11 (0.74 g, 2.64 mmol) that had been pre-cooled to 0 °C were successively
added BzCl (0.61 mL, 5.28 mmol) and DMAP (32.20 mg, 0.26 mmol), and the resulting
mixture was stirred at 0 °C for 10 min and then for another 1 h at room temperature. The
reaction mixture was concentrated and partitioned between ethyl acetate (14 mL) and water (7
mL). The aqueous layer was washed with a further portion of ethyl acetate (14 mL). The
combined organic extracts were dried over Na
2
SO , the solvent was taken down, and the
4
residue was subjected to flash chromatography on silica gel (n-hexane/ethyl acetate, 7:1) to
2
4
afford 0.98 g (97%) of compound 12 as a colourless oil; [α]
D
–27.3 (c 0.32, CHCl
3
). IR
): δ
-1 1
(
neat) υmax 2939, 1713, 1602, 1452, 1269, 1095, 1021 cm ; H NMR (400 MHz, CDCl
3

1
1
.35 (s, 3H, CH
H, J5,4 = 7.1 Hz, J4,3 = 7.1 Hz, J5,4 = 5.1 Hz, H-4), 4.65 (dd, 1H, J3,2 = 4.9 Hz, J2,1 = 3.9 Hz,
Ph), 4.73–4.78 (m, 3H, 2 × H-5, OCH Ph), 5.77 (d,
H, J2,1 = 3.9 Hz, H-1), 7.23–7.49 (m, 7H, Ph), 7.50–7.57 (m, 1H, Ph), 8.06–8.15 (m, 2H,
3
), 1.69 (s, 3H, CH
3
), 4.17 (dd, 1H, J4,3 = 7.4 Hz, J3,2 = 5.0 Hz, H-3), 4.48 (dt,
H-2), 4.67 (d, 1H, JH,H = 11.7 Hz, OCH
2
2
1
1
3
Ph); C NMR (100 MHz, CDCl
3
): δ 26.0 (CH
C-3), 78.1 (C-2), 78.4 (C-4), 104.8 (C-1), 113.8 (C
2 × CHPh), 128.5 (2 × CHPh), 129.7 (2 × CHPh), 130.2 (C
C=O). Anal. Calcd for C22 : C, 68.74; H, 6.29. Found: C, 68.79; H, 6.25.
3
), 26.6 (CH
), 127.9 (2 × CHPh), 128.0 (CHPh), 128.2
), 132.8 (CHPh), 137.2 (C ), 166.4
3 2
), 65.0 (C-5), 72.6 (OCH Ph), 77.3
(
(
(
q
i
i
24 6
H O
4
.1.7. (2S,3S)-3-(Benzyloxy)-2,4-dihydroxybutyl benzoate (14)
Compound 12 (0.78 g, 2.03 mmol) was treated with a solution of 80% TFA (3.60 mL) at 0
C, and the resulting mixture was stirred at 0 °C for 4 h. The mixture was allowed to warm to
room temperature, then was partitioned between ethyl acetate (8 mL) and water (7 mL), and
subsequently the solid NaHCO (3.36 g) was added. The insoluble material was filtered off
and the filtrate was washed with ethyl acetate (2 × 8 mL). The combined organic layers were
dried over Na SO , the solvent was removed under reduced pressure, and the residue was
purified by flash chromatography through a short column of silica gel (n-hexane/ethyl acetate,
:1) to give 0.685 g (98%) of 5-O-benzoyl-3-O-benzyl-L-lyxofuranose 13 as a colourless oil
that was used immediately in the next reaction without spectral characterization.
To a solution of 13 (0.685 g, 1.99 mmol) in a mixture of MeOH/H O (1:1, 4.60 mL) was
added NaIO (0.51 g, 2.37 mmol), and the reaction mixture was stirred at room temperature
for 1 h. Then, the mixture was diluted with CH Cl (13.50 mL), the insoluble parts were
filtered off, and the filtrate was concentrated. The residue obtained was dissolved in CH Cl
9 mL), washed with a saturated NaHCO solution (5 mL), and the aqueous layer was
extracted with another portion of CH Cl (9 mL). The combined organic phases were dried
over Na SO , the solvent was evaporated in vacuo, and the crude aldehyde (0.68 g, 1.99
mmol) was dissolved in a mixture of CH Cl /MeOH (1:4, 8.70 mL). To this solution that had
been pre-cooled to 0 °C was added NaBH (90 mg, 2.38 mmol), and the resulting mixture was
°
3
2
4
1
2
4
2
2
2
2
(
3
2
2
2
4
2
2
4
stirred at 0 °C for 10 min and then for another 30 min at room temperature. The reaction was
+
quenched by neutralization with Amberlite IR 120 (H form), the solid parts were removed by
filtration, and the filtrate was concentrated. The residue was chromatographed on silica gel (n-
hexane/ethyl acetate, 1:3) to afford 0.53 g (84%) of compound 14 as white crystals; mp 85‒86
2
2
14b
25
°
C (recrystallized from n-hexane/ethyl acetate); {[α]
D
3 D
+25.6 (c 0.34, CHCl ); lit. [α]

‒
3
30.7 (c 0.28, CHCl ) for ent-14}. IR (neat) υmax 3312, 3064, 3035, 2958, 2921, 2858, 1714,
-
1 1
1
1
4
450, 1275, 1091 cm ; H NMR (400 MHz, CD OD): δ 3.54–3.58 (m, 1H, H-3), 3.72 (dd,
3
H, J4,4 = 11.2 Hz, J4,3 = 5.3 Hz, H-4), 3.79 (dd, 1H, J4,4 = 11.4 Hz, J4,3 = 5.5 Hz, H-4), 4.04–
.08 (m, 1H, H-2), 4.29 (dd, 1H, J1,1 = 10.8 Hz, J2,1 = 5.7 Hz, H-1), 4.33 (dd, 1H, J1,1 = 10.5
2
Hz, J2,1 = 5.0 Hz, H-1), 4.57 (d, 1H, JH,H = 11.6 Hz, OCH Ph), 4.71 (d, 1H, JH,H = 11.6 Hz,
OCH Ph), 7.15–7.33 (m, 5H, Ph), 7.39–7.42 (m, 2H, Ph), 7.52–7.56 (m, 1H, Ph), 7.93–7.95
2
1
3
(
(
m, 2H, Ph); C NMR (100 MHz, CD
OCH Ph), 80.6 (C-3), 128.8 (CHPh), 129.3 (2 × CHPh), 129.4 (2 × CHPh), 129.6 (2 × CHPh),
30.7 (2 × CHPh), 131.4 (C ), 134.3 (CHPh), 139.8 (C ), 167.9 (C=O). Anal. Calcd for
: C, 68.34; H, 6.37. Found: C, 68.38; H, 6.33.
3
OD): δ 61.8 (C-4), 66.9 (C-1), 70.1 (C-2), 73.9
2
1
i
i
18 20 5
C H O
4
.1.8. [(4'S,5'S)-5'-(Benzyloxy)-2',2'-dimethyl-1',3'-dioxan-4'-yl]methyl benzoate
15)
2
To a solution of 14 (0.45 g, 1.42 mmol) in dry CH Cl (8 mL) were successively added 2,2-
(
2
dimethoxypropane (0.53 mL, 4.27 mmol) and p-TsOH (10.80 mg, 0.06 mmol), and the
reaction mixture was stirred at room temperature for 2 h. After cautious washing with a
saturated NaHCO
Na SO , the solvent was taken down, and the residue was chromatographed on silica gel (n-
hexane/ethyl acetate, 5:1) to give 0.45 g (89%) of compound 15 as white crystals mp 46.5–48
3
solution (2 × 7.70 mL), the combined organic layers were dried over
2
4
2
3
14b
25
°
C (recrystallized from n-hexane/ethyl acetate); {[α]
52.7 (c 0.26, CHCl
NMR (400 MHz, CDCl
.92 (dd, 1H, J6',6' = 12.9 Hz, J6',5' = 2.1 Hz, H-6'), 4.07 (dd, 1H, J6',6' = 12.9 Hz, J6',5' = 2.0 Hz,
H-6'), 4.27 (dt, 1H, J4',1 = 6.2 Hz, J4',1 = 6.2 Hz, J5',4' = 2.0 Hz, H-4'), 4.44 (dd, 1H, J1,1 = 11.0
Hz, J4',1 = 6.3 Hz, H-1), 4.48–4.52 (m, 2H, H-1, OCH Ph), 4.77 (d, 1H, JH,H = 12.3 Hz,
OCH Ph), 7.19–7.36 (m, 5H, Ph), 7.39–7.43 (m, 2H, Ph), 7.53–7.57 (m, 1H, Ph), 7.93–7.97
D
+50.3 (c 0.32, CHCl
3
); lit. [α]
D
-
1
1
‒
3
) for ent-15}. IR (neat) υmax 2986, 2947, 1714, 1603, 1267, 1072 cm ; H
3
3 3
): δ 1.46 (s, 3H, CH ), 1.48 (s, 3H, CH ), 3.36–3.40 (m, 1H, H-5'),
3
2
2
1
3
(
m, 2H, Ph); C NMR (100 MHz, CDCl
3
3 3
): δ 19.0 (CH ), 28.8 (CH ), 61.0 (C-6'), 64.0 (C-1),
6
9.1 (C-5'), 69.5 (C-4'), 70.8 (OCH
2
Ph), 98.8 (C ), 127.7 (CHPh), 128.0 (2 × CHPh), 128.2 (2 ×
q
CHPh), 128.3 (2 × CHPh), 129.6 (2 × CHPh), 129.9 (C
i
), 132.9 (CHPh), 137.7 (C
i
), 166.2 (C=O).
Anal. Calcd for C21 : C, 70.77; H, 6.79. Found: C, 70.81; H, 6.75.
24 5
H O
3
0,34
4
.1.9. Dimethyl (4R,5R)-2-phenyl-1,3-dioxolane-4,5-dicarboxylate (17)
To a solution of dimethyl L-tartrate (22 g, 0.15 mol) in dry benzene (220 mL) were
successively added benzaldehyde (18.3 mL, 0.18 mol) and p-TsOH (0.73 g, 3.84 mmol), and

the resulting mixture was stirred at reflux for 38.5 h using a Dean-Stark device. Then, the
reaction mixture was neutralized with Et N (0.73 mL), the solvent was removed under
3
reduced pressure, and the residue was purified by flash chromatography on silica gel (n-
hexane/ethyl acetate, 5:1) to give 32.77 g (84%) of compound 17 as white crystals mp 71–
2
D
7
30
7
1.5 °C (recrystallized from n-hexane/ethyl acetate); {[α]
–43.1 (c 0.54, MeOH); lit [α]
D
3
4
20
‒
42.0 (c 0.50, temperature and solvent not reported); lit. [α]
D
‒47.2 (c 1.00, MeOH)}. IR
-
1
1
(
neat) υmax 2958, 2909, 1751, 1429, 1236, 1214, 1102, 1079 cm ; H NMR (400 MHz,
CDCl ): δ 3.83 (s, 3H, OCH ), 3.87 (s, 3H, OCH ), 4.87 (d, 1H, J = 4.0 Hz, CH), 4.98 (d, 1H,
J = 4.0 Hz, CH), 6.14 (s, 1H, H-2), 7.39–7.41 (m, 3H, Ph), 7.57–7.59 (m, 2H, Ph); C NMR
100 MHz, CDCl ): δ 52.9 (2 × OCH ), 77.2 (CH), 77.4 (CH), 106.7 (C-2), 127.2 (2 × CHPh),
28.4 (2 × CHPh), 130.0 (CHPh), 135.2 (C ), 169.4 (C=O), 170.0 (C=O). Anal. Calcd for
: C, 58.64; H, 5.30. Found: C, 58.60; H, 5.35.
3
3
3
1
3
(
3
3
1
i
13 14 6
C H O
3
0,34
4
.1.10. (2S,3S)-3-(Benzyloxy)butane-1,2,4-triol (18)
To a suspension of LiAlH
4
(17.60 g, 0.46 mol) in a mixture of Et
2
O (195 mL) and CH
2
Cl
2
(
180 mL), pre-cooled to 0 °C, was added compound 17 (32.43 g, 0.12 mol), and the mixture
was stirred at 0 °C for 40 min. A solution of AlCl (52.90 g, 0.40 mol) in dry Et O (195 mL)
3
2
was added, and the reaction mixture was stirred at reflux for 2.5 h. After cooling to room
temperature and cautious addition of water (111 mL) and a 15% NaOH solution (240 mL), the
solids parts were filtered through a small pad of Celite. The aqueous phase was washed with
ethyl acetate (2 × 250 mL), the combined organic layers were dried over Na
2
SO , the solvent
4
was evaporated, and the residue was flash-chromatographed through a short column of silica
gel to furnish 20.7 g (80%) of compound 18 as white crystals; mp 73–74.5 °C (recrystallized
2
5
34
22
30
from ethyl acetate); {[α]
not reported}. IR (neat) υmax 3250, 2969, 2940, 1449, 1364, 1202, 1036 cm ; H NMR (400
MHz, CD OD): δ 3.56 (dt, 1H, J2,1 = 5.3 Hz, J2,1 = 5.3 Hz, J3,2 = 3.9 Hz, H-2), 3.59 (dd, 1H,
4,4 = 11.1 Hz, J4,3 = 6.5 Hz, H-4), 3.64 (dd, 1H, J4,4 = 11.1 Hz, J4,3 = 5.1 Hz, H-4), 3.69 (dd,
D
+14.7 (c 0.58, MeOH); lit [α]
D
+15.7 (c 1.00, MeOH); lit [α]
D
-1 1
3
J
1
H, J1,1 = 11.6 Hz, J2,1 = 5.4 Hz, H-1), 3.76–3.80 (m, 2H, H-1, H-3), 4.62 (d, 1H, JH,H = 11.5
1
3
Hz, OCH
2
Ph), 4.73 (d, 1H, JH,H = 11.5 Hz, OCH
OD): δ 62.1 (C-1), 64.1 (C-4), 73.1 (C-3), 74.1 (OCH
16 4
). Anal. Calcd for C11H O
2
Ph), 7.24–7.40 (m, 5H, Ph); C NMR (100
Ph), 81.3 (C-2), 128.7
: C, 62.25;
MHz, CD
3
2
(
CHPh), 129.1 (2 × CHPh), 129.3 (2 × CHPh), 140.1 (C
i
H, 7.60. Found: C, 62.21; H, 7.65.

3
0
4
.1.11. [(4'S,5'S)-5'-(Benzyloxy)-2',2'-dimethyl-1',3'-dioxan-4'-yl]methanol (16)
Modification of 15 into 16:
CO (24.4 mg, 0.18 mmol) was added to a solution of 15 (0.21 g, 0.59 mmol) in MeOH (6
mL), pre-cooled to 0 °C, and the reaction mixture was stirred at 0 °C for 15 min and then at
room temperature for another 4 h. After dilution with Et O (18 mL), the solid Na SO was
K
2
3
2
2
4
added, and salts were removed by filtration. Evaporation of solvent and chromatography of
the residue on silica gel (n-hexane/ethyl acetate, 1:1) afforded 0.14 g (95%) of compound 16
as white crystals.
Modification of 18 into 16:
To a solution of triol 18 (20.47 g, 96.5 mmol) in dry DMF (235 mL), pre-cooled to –15 °C,
were successively added PPTS (1.94 g, 7.72 mmol) and 2-methoxypropene (11.1 mL, 0.12
mol) dissolved in dry DMF (47 mL). The reaction mixture was stirred at –15 °C for 5 h and
then at room temperature for another 1 h. The reaction was quenched by neutralization with
Et
solution (250 mL). The organic layer was dried over Na
the residue was chromatographed on silica gel (n-hexane/ethyl acetate, 3:1) to give 5.84 g
3
N, subsequently diluted with ethyl acetate (100 mL) and washed with a saturated NaCl
2
SO , the solvent was evaporated, and
4
3
0
(
(
(
24%) of the require alcohol 16 together with a barely separable mixture of compounds:
3
0
2S)-2-(benzyloxy)-2-[(4'S)-2',2'-dimethyl-1',3'-dioxolan-4'-yl]ethanol
and
(5S,6S)-6-
3
0
benzyloxy)-2,2-dimethyl-1,3-dioxepan-5-ol (16.54 g, 68%). Repeated chromatography on
silica gel (n-hexane/ethyl acetate, 3:1) afforded both aforementioned derivatives in pure form
as analytical samples. Their physical and spectroscopic properties were in agreement with
3
0
literature data.
2
5
Alcohol 16: mp 60–61 °C (recrystallized from n-hexane/ethyl acetate); {[α]
D
+64.7 (c 0.30,
3
0
14b
23
CHCl
3
); lit ([α]
D
+64.0 (c 0.30, temperature and solvent not reporte); lit. [α]
D
‒69.4 (c
-
0
.32, CHCl
1
3
) for ent-16}. IR (neat) υmax 3502, 2992, 2960, 2940, 2867, 1455, 1382, 1170 cm
H NMR (400 MHz, CDCl ): δ 1.45 (s, 3H, CH ), 1.47 (s, 3H, CH
.30–3.31 (m, 1H, H-5'), 3.60–3.63 (m, 1H, H-1), 3.64 (dd, 1H, J1,1 = 11.4 Hz, J4',1 = 6.7 Hz,
H-1), 3.90 (dd, 1H, J6',6' = 12.9 Hz, J6',5' = 2.2 Hz, H-6'), 3.96 (ddd, 1H, J4',1 = 6.8 Hz, J4',1
.8 Hz, J5',4' = 2.2 Hz, H-4'), 4.02 (dd, 1H, J6',6' = 12.9 Hz, J6',5' = 2.2 Hz, H-6'), 4.43 (d, 1H,
1
;
3
3
3
), 2.02 (br s, 1H, OH),
3
=
4
1
3
J
H,H = 12.3 Hz, OCH
NMR (100 MHz, CDCl
OCH Ph), 71.6 (C-4'), 98.8 (C
: C, 66.65; H, 7.99. Found: C, 66.61; H, 8.04.
2
Ph), 4.75 (d, 1H, JH,H = 12.3 Hz, OCH
): δ 19.1 (CH ), 28.8 (CH ), 61.1 (C-6'), 62.8 (C-1), 70.0 (C-5'), 70.7
), 127.9 (3 × CHPh), 128.4 (2 × CHPh), 137.8 (C ). Anal. Calcd
2
Ph), 7.28–7.35 (m, 5H, Ph); C
3
3
3
(
2
q
i
20 4
for C14H O

4
.1.12. Ethyl (E)-3-[(4'S,5'S)-5'-(benzyloxy)-2',2'-dimethyl-1',3'-dioxan-4'-
yl]acrylate [(E)-19] and ethyl (Z)-3-[(4'S,5'S)-5'-(benzyloxy)-2',2'-dimethyl-1',3'-dioxan-
'-yl]acrylate [(Z)-20]
4
o-Iodoxybenzoic acid (15.94 g, 56.90 mmol) was added to a solution of 16 (5.74 g, 22.77
mmol) in MeCN (207 mL) and the resulting suspension was stirred and heated to reflux for 1
h. After cooling to room temperature, the insoluble material was filtered off, the solvent was
removed, and the crude aldehyde was used immediately in the subsequent reaction without
further purification.
To a suspension of NaH (0.74 g, 31 mmol, 60% dispersion in mineral oil) in dry THF (48.50
mL) that was pre-cooled to –10 °C was added dropwise ethyl 2-(diethoxyphosphoryl)acetate
(
5 mL, 25 mmol), and the mixture was stirred for 25 min at 0 °C. To this mixture, we added a
solution of the crude aldehyde (5.70 g, 22.77 mmol) in dry THF (9.80 mL) at 0 °C. After
stirring for 1 h at 0 °C, the suspension was poured into a saturated NH Cl solution (17.40 mL)
and extracted with ethyl acetate (2 × 25 mL). The combined organic layers were dried over
Na SO , the solvent was taken down, and the residue was chromatographed on silica gel (n-
hexane/ethyl acetate, 5:1) to afford 5.91 g (81%) of (E)-19 and 0.875 g (12%) of (Z)-20.
4
2
4
2
D
3
14b
22
D
Isomer (E)-19: colourless oil; {[α]
CHCl ) for ent-(E)-19}. IR (neat) υmax 2988, 2939, 2869, 1714, 1662, 1368, 1261 cm ; H
NMR (400 MHz, CDCl ): δ 1.30 (t, 3H, J = 7.1 Hz, CH ), 1.46 (s, 3H, CH ), 1.50 (s, 3H,
CH ), 3.31–3.32 (m, 1H, H-5'), 3.95 (dd, 1H, J6',6' = 12.8 Hz, J6',5' = 2.3 Hz, H-6'), 4.01 (dd,
H, J6',6' = 12.8 Hz, J6',5' = 2.2 Hz, H-6'), 4.22 (q, 2H, J = 7.1 Hz, CH ), 4.50 (d, 1H, JH,H
2.6 Hz, OCH Ph), 4.57 (td, 1H, J4',3 = 4.3 Hz, J5',4' = 2.1, J4',2 = 2.1 Hz, H-4'), 4.70 (d, 1H,
H,H = 12.6 Hz, OCH Ph), 6.13 (dd, 1H, J3,2 = 15.7 Hz, J4',2 = 1.8 Hz, H-2), 6.91 (dd, 1H, J3,2
15.7 Hz, J4',3 = 4.3 Hz, H-3), 7.26–7.32 (m, 5H, Ph); C NMR (100 MHz, CDCl
CH ), 19.1 (CH ), 28.8 (CH ), 60.3 (CH ), 61.6 (C-6'), 2 × 70.9 (C-4', C-5'), 71.2 (OCH
9.0 (C ), 121.8 (C-2), 127.8 (CHPh), 128.0 (2 × CHPh), 128.3 (2 × CHPh), 137.7 (C ), 144.5
: C, 67.48; H, 7.55. Found: C, 67.43; H, 7.59.
Isomer (Z)-20: white crystals, mp 41‒42 °C (recrystallized from n-hexane/ethyl acetate);
‒11.4 (c 0.36, CHCl
3
); lit. [α]
+12.3 (c 0.26,
-1
1
3
3
3
3
3
1
1
2
=
2
J
2
1
3
=
3
): δ 14.3
Ph),
(
3
3
3
2
2
9
q
i
(
C-3), 166.2 (C=O). Anal. Calcd for C18
H
24
O
5
2
3
14b
22
{
υ
3
1
4
[α]
max 2989, 2958, 2889, 1706, 1645, 1371, 1185 cm ; H NMR (400 MHz, CDCl
H, J = 7.1 Hz, CH ), 1.48 (s, 3H, CH ), 1.49 (s, 3H, CH ), 3.58–3.60 (m, 1H, H-5'), 3.96 (dd,
H, J6',6' = 13.0 Hz, J6',5' = 2.1Hz, H-6'), 4.01 (dd, 1H, J6',6' = 12.9 Hz, J6',5' = 2.1 Hz, H-6'),
.09 (q, 2H, J = 7.1 Hz, CH ), 4.45 (d, 1H, JH,H = 12.4 Hz, OCH Ph), 4.67 (d, 1H, JH,H = 12.4
D
+186.0 (c 0.33, CHCl
3
); lit. [α]
D
–203.0 (c 0.28, CHCl
3
) for ent-(Z)-20}. IR (neat)
-1 1
3
): δ 1.26 (t,
3
3
3
2
2

Hz, OCH
2
Ph), 5.50–5.53 (m, 1H, H-4'), 5.80 (dd, 1H, J3,2 = 11.8 Hz, J4',2 = 1.5 Hz, H-2), 6.39
13
(
dd, 1H, J3,2 = 11.8 Hz, J4',3 = 6.8 Hz, H-3), 7.23–7.30 (m, 5H, Ph); C NMR (100 MHz,
CDCl
'), 71.4 (OCH
C-3), 165.7 (C=O). Anal. Calcd for C18
3
): δ 14.1 (CH
3
), 19.1 (CH
3
), 29.2 (CH
3
), 60.2 (CH
2
), 61.6 (C-6'), 69.4 (C-4'), 71.2 (C-
), 148.3
: C, 67.48; H, 7.55. Found: C, 67.53; H, 7.52.
5
2
Ph), 99.8 (C
q
), 119.6 (C-2), 127.6 (CHPh), 128.1 (4 × CHPh), 138.0 (C
i
(
H
24
O
5
4
1
.1.13. (E)-3-[(4'S,5'S)-5'-(Benzyloxy)-2',2'-dimethyl-1',3'-dioxan-4'-yl]prop-2-en-
-ol (21)
Ester (E)-19 (5.85 g, 18.26 mmol) was dissolved in dry CH
2
Cl (82.50 mL), and the resulting
2
solution was cooled to –50 °C. Diisobutylaluminum hydride (45.60 mL, 54.80 mmol, 1.2 M
toluene solution) was added dropwise, and the mixture was then stirred for 30 min at –30 °C
before quenching with MeOH (18 mL). After warming to room temperature, the mixture was
poured into a 30% K/Na tartrate solution (385 mL) with vigorous stirring over 1.5 h at room
temperature. The separated aqueous phase was extracted with CH
combined organic layers were dried over Na SO , stripped of solvent, and the residue was
subjected to flash chromatography on silica gel (n-hexane/ethyl acetate, 2:1) to furnish 4.73 g
2
Cl
2
(3 × 215 mL). The
2
4
(
93%) of compound 21 as white crystals; mp 41–43 °C (recrystallized from n-hexane/ethyl
2
6
14b
23
acetate); {[α]
D
+38.7 (c 0.54, CHCl
3
); lit. [α]
D
–31.9 (c 0.26, CHCl
3
) for ent-21}. IR
): δ 1.47 (s,
), 3.20–3.22 (m, 1H, H-5'), 3.94 (dd, 1H, J6',6' = 12.8 Hz, J6',5' = 2.2
Hz, H-6'), 4.00 (dd, 1H, J6',6' = 12.8 Hz, J6',5' = 2.2 Hz, H-6'), 4.09–4.17 (m, 2H, 2 × H-1), 4.41
dd, 1H, J4',3 = 4.5 Hz, J5',4' = 2.2 Hz, H-4'), 4.50 (d, 1H, JH,H = 12.6 Hz, OCH Ph), 4.73 (d,
H, JH,H = 12.6 Hz, OCH Ph), 5.83–5.93 (m, 2H, H-2, H-3),7.26–7.36 (m, 5H, Ph); C NMR
100 MHz, CDCl ): δ 19.1 (CH ), 29.1 (CH ), 61.7 (C-6'), 63.0 (C-1), 71.3 (OCH Ph), 71.8
C-5'), 72.2 (C-4'), 98.8 (C ), 127.7 (CHPh), 128.1 (2 × CHPh), 128.2 (2 × CHPh), 128.7 (C-2 or
). Anal. Calcd for C16 : C, 69.04; H, 7.97. Found: C,
-
1 1
(
neat) υmax 3452, 2993, 2866, 1389, 1198, 1065 cm ; H NMR (400 MHz, CDCl
3
3
H, CH ), 1.48 (s, 3H, CH
3
3
(
2
1
3
1
2
(
(
3
3
3
2
q
C-3), 131.8 (C-2 or C-3), 138.1 (C
i
22
H O
4
6
9.08; H, 7.93.
4
.1.14. (4S,5S)-5-(Benzyloxy)-2,2-dimethyl-4-[(E)-3'-thiocyanatoprop-1'-en-1'-yl]-1,3-
dioxan (22)
2 2
To a solution of 21 (1.81 g, 6.50 mmol) in dry CH Cl (78.60 mL) were successively added
Et
3
N (1.37 mL, 9.75 mmol) and methanesulfonyl chloride (0.75 mL, 9.75 mmol) at 0 °C. The
resulting mixture was stirred for 30 min at 0 °C and then for a further 10 min at room

temperature. After evaporating of the solvent, the residue was diluted with Et
2
O (20 mL), the
insoluble parts were filtered off and washed with Et O. Removal of the solvent afforded a
2
mesylate that was used in the next reaction step without purification.
KSCN (1.07 g, 11.05 mmol) was added to a solution of the crude mesylate (2.32 g, 6.50
mmol) in dry MeCN (55.50 mL) at 0 °C. The resulting mixture was stirred for 15 min at 0 °C
and then for another 6.5 h at room temperature before evaporating of the solvent. To such
residue, Et
2
O (20 mL) was added producing salts, which were removed by filtration. The
filtrate was concentrated to provide an orange oil, which was chromatographed on silica gel
(
n-hexane/ethyl acetate, 5:1) to give 1.87 g (90%) of compound 22 as white crystals; mp 49–
2
7
14b
23
5
–
1
0 °C (recrystallized from n-hexane/ethyl acetate); {[α]
49.6 (c 0.26, CHCl ) for ent-22}. IR (neat) υmax 2988, 2940, 2866, 2150, 1453, 1377, 1193,
078 cm ; H NMR (400 MHz, CDCl ): δ 1.47 (s, 3H, CH ), 1.48 (s, 3H, CH ), 3.25 (q, 1H,
6,5 = 2.2 Hz, J6,5 = 2.2 Hz, J5,4 = 2.2 Hz, H-5), 3.55 (dd, 1H, J3',3' = 12.7 Hz, J3',2' = 5.5 Hz, H-
'), 3.61 (dd, 1H, J3',3' = 12.9 Hz, J3',2' = 7.3 Hz, H-3'), 3.92 (dd, 1H, J6,6 = 12.9 Hz, J6,5 = 2.2
Hz, H-6), 3.99 (dd, 1H, J6,6 = 12.9 Hz, J6,5 = 2.1 Hz, H-6), 4.47 (dd, 1H, J4,1' = 4.8 Hz, J5,4
D
3 D
+42.6 (c 0.38, CHCl ); lit. [α]
3
-1 1
3
3
3
J
3
=
2
5
.0 Hz, H-4), 4.54 (d, 1H, JH,H = 12.5 Hz, OCH
.83–5.95 (m, 2H, H-1', H-2'), 7.26–7.36 (m, 5H, Ph); C NMR (100 MHz, CDCl
), 29.0 (CH ), 35.8 (C-3'), 61.5 (C-6), 71.3 (OCH Ph), 71.4 (C-4), 71.5 (C-5), 98.9 (C
11.8 (SCN), 124.5 (C-2'), 127.7 (CHPh), 128.0 (2 × CHPh), 128.3 (2 × CHPh), 134.5 (C-1'),
37.9 (C ). Anal. Calcd for C17 S: C, 63.92; H, 6.63; N, 4.39. Found: C, 63.90; H, 6.59;
2
Ph), 4.71 (d, 1H, JH,H = 12.5 Hz, OCH
2
Ph),
): δ 19.1
),
1
3
3
(
CH
3
3
2
q
1
1
i
H21NO
3
N, 4.43.
4
.1.15.
(4S,5S)-5-(Benzyloxy)-4-[(1'S)-1'-isothiocyanatoallyl]-2,2-dimethyl-1,3-dioxan
(
(
24) and (4S,5S)-5-(benzyloxy)-4-[(1'R)-1'-isothiocyanatoallyl]-2,2-dimethyl-1,3-dioxan
25)
4
.1.15.1. Conventional method
A solution of thiocyanate 22 (0.10 g, 0.31 mmol) in n-heptane (2.6 mL) was stirred and
heated under a nitrogen atmosphere (for the reaction times and temperatures, see Table 1).
After cooling to room temperature, the solvent was removed under reduced pressure, and the
residue was chromatographed on silica gel (n-hexane/ethyl acetate, 5:1) to give the
corresponding isothiocyanates 24 and 25 as colourless oils (for the combined yields, see Table
1
).

Requiring a greater amount of the pure rearranged products 24 and 25, they were obtained on
a multi-gram scale by the conventional method in n-heptane at 90 °C.
4
.1.15.2. Microwave-assisted synthesis
Thiocyanate 22 (0.10 g, 0.31 mmol) was weighed in a 10-mL glass pressure microwave tube
equipped with a magnetic stirbar. n-Heptane (2.60 mL) was added, the tube was closed with a
silicon septum, and the resulting mixture was subjected to microwave irradiation (for the
temperatures and reaction times, see Table 1). Evaporating of the solvent and chromatography
on silica gel (n-hexane/ethyl acetate, 5:1) gave isothiocyanates 24 and 25 (for the combined
yields, see Table 1).
2
D
5
14b
23
D
Diastereoisomer 24: {[α]
+63.1 (c 0.44, CHCl
3
); lit. [α]
3
–61.4 (c 0.28, CHCl ) for ent-
-1
1
2
4}. IR (neat) υmax 2990, 2870, 2170, 2087, 1454, 1377, 1197 cm ; H NMR (400 MHz,
CDCl
3
): δ 1.40 (s, 3H, CH
.4 H, J5,4 = 2.0 Hz, H-4), 3.89 (dd, 1H, J6,6 = 13.1 Hz, J6,5 = 2.0 Hz, H-6), 4.07 (dd, 1H, J6,6
3.1 Hz, J6,5 = 2.0 Hz, H-6), 4.59 (d, 1H, JH,H = 11.7 Hz, OCH
.75 (d, 1H, JH,H = 11.6 Hz, OCH Ph), 5.28 (dd, 1H, J3',2' = 10.4 Hz, J3',1' = 1.5 Hz, J3',3' = 0.7
3
), 1.42 (s, 3H, CH
3
), 3.48–3.49 (m, 1H, H-5), 3.79 (dd, 1H, J4,1'
=
9
1
4
=
2
Ph), 4.61–4.65 (m, 1H, H-1'),
2
Hz, H-3'cis), 5.44 (dd, 1H, J3',2' = 17.0 Hz, J3',1' = 1.7 Hz, J3',3' = 0.7 Hz, H-3'trans), 5.95 (dd, 1H,
1
3
J
3',2' = 17.0 Hz, J3',2' = 10.4 Hz, J2',1' = 4.7 Hz, H-2'), 7.29–7.42 (m, 5H, Ph); C NMR (100
MHz, CDCl ): δ 19.0 (CH ), 28.8 (CH ), 58.1 (C-1'), 61.0 (C-6), 69.1 (C-5), 71.4 (OCH Ph),
4.0 (C-4), 99.2 (C ), 117.0 (C-3'), 127.9 (CHPh), 128.2 (2 × CHPh), 128.5 (2 × CHPh), 132.7
C-2'), 133.9 (NCS), 137.5 (C ). Anal. Calcd for C17 S: C, 63.92; H, 6.63; N, 4.39.
Found: C, 63.96; H, 6.59; N, 4.42.
3
3
3
2
7
q
(
i
H21NO
3
2
5
14b
25
Diastereoisomer 25: {[α]
5}. IR (neat) υmax 3030, 2990, 2873, 2041, 1454, 1376, 1197, 1078 cm ; H NMR (400
MHz, CDCl ): δ 1.49 (s, 3H, CH ), 1.50 (s, 3H, CH ), 3.25–3.26 (m, 1H, H-5), 3.82 (dd, 1H,
4,1' = 9.1 Hz, J5,4 = 2.0 Hz, H-4), 3.87 (dd, 1H, J6,6 = 13.2 Hz, J6,5 = 2.2 Hz, H-6), 4.11 (dd,
H, J6,6 = 13.2 Hz, J6,5 = 2.1 Hz, H-6), 4.37 (d, 1H, JH,H = 11.8 Hz, OCH Ph), 4.61–4.65 (m,
H, H-1'), 4.72 (d, 1H, JH,H = 11.8 Hz, OCH Ph), 5.23–5.26 (m, 1H, H-3'cis), 5.40–5.45 (m,
H, H-3'trans), 5.53 (ddd, 1H, J3',2' = 16.8 Hz, J3',2' = 9.9 Hz, J2',1' = 5.6 Hz, H-2'), 7.28–7.37 (m,
D
+42.0 (c 0.20, CHCl
3
); lit. [α]
D
3
–41.9 (c 0.32, CHCl ) for ent-
-1
1
2
3
3
3
J
1
1
1
5
2
2
1
3
H, Ph); C NMR (100 MHz, CDCl
3
): δ 18.9 (CH
), 119.6 (C-3'), 127.9 (3 × CHPh), 128.5 (2 ×
). Anal. Calcd for C17 S: C, 63.92; H, 6.63;
3 3
), 28.7 (CH ), 60.5 (C-1'), 60.7 (C-6), 69.2
(
C-5), 70.5 (OCH
2
Ph), 74.6 (C-4), 99.6 (C
q
CHPh), 130.3 (C-2'), 135.9 (NCS), 137.5 (C
i
H21NO
3
N, 4.39. Found: C, 63.88; H, 6.67; N, 4.33.