Total synthesis and the anticancer activity of (+)-spisulosine

Article abstract of DOI:10.1016/j.carres.2016.09.010

The total synthesis of the anticancer agent (+)-spisulosine has been accomplished. The strategy involved a substrate-controlled aza-Claisen rearrangement to establish the erythro-configured amino-alcohol motif followed by deoxygenation to create a methyl

Full text of DOI:10.1016/j.carres.2016.09.010

Carbohydrate Research 435 (2016) 26e36
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Carbohydrate Research
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Total synthesis and the anticancer activity of (þ)-spisulosine
,
*
a
ꢀ
ꢁ a
ꢁ
ꢁ a
ꢁ a
Milica Fabisíkova , Miroslava Martinkova , Simona Hirkova , Jozef Gonda ,
ꢁ b
€
ꢁ b
Martina Bago Pilatova , Gabriela Gonciova
a
ꢀ
ꢁ
ꢀ
Department of Organic Chemistry, Institute of Chemical Sciences, P.J. Safarik University, Moyzesova 11, 040 01 Kosice, Slovak Republic
b
ꢀ
ꢁ
ꢀ
Department of Pharmacology, Faculty of Medicine, P.J. Safarik University, SNP 1, 040 66 Kosice, Slovak Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
The total synthesis of the anticancer agent (þ)-spisulosine has been accomplished. The strategy involved
a substrate-controlled aza-Claisen rearrangement to establish the erythro-configured amino-alcohol
motif followed by deoxygenation to create a methyl side-chain. Subsequent Wittig olefination then
permitted the construction of the carbon backbone of the target molecule. To investigate the anti-
proliferative effect of 1, its biological profile was examined on a panel of 6 human malignant cell lines
and demonstrated the significant anticancer activity of 1 on at least five of the evaluated lines with
Received 22 July 2016
Received in revised form
25 August 2016
Accepted 20 September 2016
Available online 21 September 2016
IC50 < 1 mM (MCF-7, HTC-116, Caco-2, Jurkat and HeLa).
Keywords:
© 2016 Elsevier Ltd. All rights reserved.
1-deoxysphingoid bases
Spisulosine
[3,3]-heterosigmatropic rearrangement
Stereoselective synthesis
Antiproliferative activity
1. Introduction
found to reduce prostate tumor cell growth via de novo synthesis of
ceramide and atypical protein kinase C (PKC ) activation [8]. Its
z
Simple saturated 1-deoxysphingoid bases of marine origin,
represented by spisulosine 1 [1], xestoaminol C (2) [2], its 3-epimer
3 [3], clavaminols [4], the general structure of which is illustrated
by clavaminol A (4) [4a] (Fig. 1), and related analogues have become
an attractive and timely target for total synthesis due to their
impressive biological activities as well as their simple, but unique
structures possessing an erythro- or threo-configured amino
alcohol moiety.
truncated analogue xestoaminol C (2) was discovered indepen-
dently by two research groups. The first of these isolated 2 from the
Fijian sponge Xestospongia sp [2a] while the second group obtained
it from the tunicate Pseudodistoma obscurum [2b]. Xestoaminol C
(2) exhibits activity against reverse transcriptase [2] as well as
remarkable cytotoxic/antiproliferative potency in several human
cancer cells A-549 [5], HT-29 [5], MeL-28 [5], DU-145 [5] and SHG-
44 [9]. It should be noted that 1 was found to possess higher in vitro
anticancer activity than xestoaminol C. In 2014, Keyzers and co-
workers [3] reported the discovery of 3-epi-xestoaminol C (3), an
effective antimicrobial compound present in the New Zealand
brown alga Xiphophora chondrophylla. The aforementioned bases,
especially spisulosine have stimulated a number of synthetic efforts
and several elegant and efficient approaches towards 1 have been
developed. The chemistry of 1 dates back to 1957 when this
molecule was synthesized for the first time by Prostenik and
Spisulosine 1 was first isolated from the clam Spisula polynyma
(syn. Mactrometris polynyma) [1] and displayed very strong cyto-
toxic activity on both leukaemia and solid cancer cells with IC50
ꢁ
values down to the nanomolar range [1,5]. Padron and co-workers
further revealed that 1 behaved as a selective CK1ε inhibitor and
demonstrated its remarkable antiproliferative potency on several
cancer cells (HBL-100, HeLa, SW1573, T-47D and WiDr) [6]. Bittman
and co-workers have found that spisulosine selectively blocks
sphingosine kinase (SphK1) to induce its ubiquitin-proteasomal
degradation in human pulmonary arterial smooth muscle cells
ꢁ
Alaupovic [10] before its isolation from the natural source [1]. To
date, several total syntheses of 1 in the form of its free base and/or
its HCl salt [1,5,7,9,11] and N-Boc-derivative [11a,11c,12] have been
reported, together with one construction of racemic ( )-spisulosine
[13]. Elaborated strategies have garnered both the Chiron approach
[1,5,7,9,11a-c,11i-j,12] as well as asymmetric methods [11d-11h].
Thus, Reinhart and co-workers [1b], Huangʼs group [9], Delgado
(PASMC) with an IC50 of 7.1 0.75 mM [7]. In addition,1 significantly
inhibits DNA synthesis in PASMC [7]. Spisulosine has also been
* Corresponding author.
ꢁ
ꢁ
and co-workers [11i], Padronʼs group [11j], and Ghosal and Shaw
E-mail address: miroslava.martinkova@upjs.sk (M. Martinkova).
http://dx.doi.org/10.1016/j.carres.2016.09.010
0008-6215/© 2016 Elsevier Ltd. All rights reserved.

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M. Fabisíkova et al. / Carbohydrate Research 435 (2016) 26e36
27
Fig. 1. Structures of the natural 1-deoxysphingoid bases.
Fig. 2. Reported approaches to spisulosine 1.
[11c] have commenced their syntheses of spisulosine 1 from the
alanine and serine scaffolds, namely -alanine methyl ester hy-
drochloride, -alanine, N-Boc- -alaninal, benzyl (S)-2-(dibenzyla-
mino)propanoate, (S)-Garnerʼs aldehyde, respectively, in which the
addition of diverse organometallic species was employed as the key
and Miyashitaʼs boron-directed C-2 regioselective azidolysis. Dau-
ban and co-workers [11e] have applied a copper-catalyzed intra-
molecular alkene aziridination of sulfamates and SN2 ring-opening
of the generated bicyclic product to provide the advanced inter-
mediate bearing two required stereocentre. Kumarʼs group [11h]
L
L
L
ꢁ
step. As well, approaches of Galvezʼs [11a] and Chemlaʼs groups
strategy has used a L-proline-catalyzed a-amination of propio-
[11b] have relied on the diastereoselective addition of these
aforementioned reagents to the N-benzylimine derived from 2,3-O-
isopropylidene-D-glyceraldehyde and N-tert-butylsulfinyl imine,
naldehyde and subsequent indium-mediated allylation, assembling
the truncated carbon fragment possessing anti-amino alcohol unit
enatio- and diastereoselectively. Our work (vide infra) is based on a
[3,3]-heterosigmatropic rearrangement to install the required CeN
bond while a Wittig reaction allowed incorporation of the long
chain (Fig. 2).
On the other hand, spisulosine truncated analogue xestoaminol
C [5,9,14] as well as its 3-epimer 3 [14a,14e,15] have received less
attention. It should be noted that ent-1 [11i] and (2S,3S)-stereo-
isomer [11d,11i], together with its (2R,3R)-antipode [11i] have been
synthesized. Delgadoʼs group [11i] evaluated 1 and all of its ste-
reoisomers for in vitro cytotoxicity against the cancer MDA-MB-
468 cell line with CC50 levels in the micromolar range. In 2011,
Dauban and co-workers [11e] communicated their preparation of
the 3-fluoro analogue of spisulosine, (2S,3R)-3-fluorooctadecan-2-
respectively. Sutherland and co-workers [11g] have developed a
MOM ether-directed palladium(II)-catalyzed Overman rearrange-
ment of an allylic trichloroacetimidate, which was prepared from
heptadec-1-ene, to create an erythro-configured amino alcohol
foldamer. Wangʼs group [11f] has reported a synthesis of 1 from
propionaldehyde, which utilized a highly anti-selective asymmetric
Henry reaction to build up the carbon backbone with both ster-
eogenic centres. Pandaʼs group [11d] construction of spisulosine
from hexadecan-1-ol has employed the Sharpless asymmetric
dihydroxylation and SN2 displacement with a nitrogen nucleophile
(azide) as the crucial transformations. Their alternative route from
(E)-octadec-2-en-1-ol has involved the asymmetric epoxidation
Scheme 1. Retrosynthetic analysis of spisulosine 1.

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M. Fabisíkova et al. / Carbohydrate Research 435 (2016) 26e36
28
Scheme 2. Reagents and conditions: (a) Ref. [19]; (b) BnBr, Ag2O, CH2Cl2, rt; (c) LiAlH4, THF, rt; (d) (i) IBX, MeCN, reflux; (ii) Ph3P ¼ CHCO2Et, CH2Cl2, rt; (e) DIBAl-H, CH2Cl2, ꢀ50 ꢁC;
(f) (i) MsCl, Et3N, CH2Cl2, 0 ꢁC/rt; (ii) KSCN, MeCN, 0 ꢁC/rt; (g) Table 1.
Table 1
[3,3]-Sigmatropic rearrangement of thiocyanate 8.
Entry
Thiocyanate
Conditionsa
MW, 70 ꢁC
Time (h)
Ratiob 14:15
Yieldc (%)
Yieldd (%)
1
2
3
4
5
6
7
8
8
8
8
8
8
8
1
21
1
4
1
78:22
70:30
56:44
56:44
83:17
80:20
58:42
64
67
73
67
65
68
65
34
32
25
32
33
30
25
D
, 70 ꢁC
MW, 90 ꢁC
MW, 90 ꢁC
D
D
D
, 90 ꢁC
, 90 ꢁC
, 90 ꢁC
2
17
a
In n-heptane.
b
c
Ratio in the crude reaction mixtures. Determined by 1H NMR spectroscopy.
Isolated combined yields.
d
Isolated yields of thiocyanate 8.
amine, and screened this molecule along with 1 for anticancer ac-
tivity on KB, HCT-116 and HL-60 cell lines. Cytotoxic activity of
spisulosine was confirmed in all cell lines, with IC50 values in the
nanomolar range. The aforementioned fluoro derivative proved
completely inactive even at micromolar concentrations [11e].
Recently, we reported a novel approach to the total synthesis of
heterogeneously configured deoxysphingoid bases employing a
diastereoselective substrate-controlled aza-Claisen rearrangement
[16]. In this paper, we would like to show an extension of this
methodology for the construction of the known cytotoxic marine
reduction of 11 (LiAlH4, THF) [18b] resulted in the formation of 9
[18] in 89% yield. To continue the synthesis, a one-pot IBX oxida-
tion/Wittig olefination of 9 furnished a mixture of a,b-unsaturated
Table 2
Crystal data and structure refinement parameters for compound 14.
14
Empirical formula
Formula weight
C17H21NO3S
319.41
Temperature, T (K)
173(2)
Wavelength,
l
(Å)
0.71073
product 1 starting from D-isoascorbic acid as the chiral template.
Crystal system
Space group
Orthorhombic
P212121
Unit cell dimensions
a (Å)
2. Results and discussion
8.4431(3)
b (Å)
11.1105(5)
2.1. Chemistry
c (Å)
17.9408(8)
1682.97(12)
V (Å3)
Formula per unit cell, Z
4
The retrosynthetic plan is outlined in Scheme 1. For spisulosine
1, disconnection of the carbon framework led to the known phos-
phonium salt 5 [17] and the polar scaffold 6 with the desired D-
Dcalcd (g/cm3)
1.261
0.204
680
Absorption coefficient,
F(0 0 0)
m
(mmꢀ1
)
Crystal size (mm)
q
0.46 ꢂ 0.37 ꢂ 0.27
2.919e26.487
ꢀ10 ꢃ h ꢃ 10
ꢀ13 ꢃ k ꢃ 11
ꢀ22 ꢃ l ꢃ 22
3474(0.0288)
Analytical
0.955 and 0.906
Full-matrix least-squares on F2
3474/0/201
erythro-configured amino alcohol unit. The deoxygenation at C-1
would be required, and it could be accomplished en route to 6 from
the pivotal compound 7. The aza-Claisen rearrangement of the
allylic substrate 8 was expected to create a new CeN bond. The
Range for data collection (o)
Index ranges
thiocyanate 8 was built up from protected
derived from -isoascorbic acid.
As shown in Scheme 2, our investigation commenced with the
D
-erythritol 9 [18]
Independent reflections (Rint)
Absorption correction
Max. and min. transmission
Refinement method
D
known ester 10 which was prepared on a multi-gram scale from
(ꢀ)-isoascorbic acid adopting the known protocol.
D-
Data/restraints/parameters
Goodness-of-fit on F2
1.058
Final R indices [I > 2
R indices (all data) Flack x parameter
Largest diff. peak and hole (e/Åꢀ3
s(I)]
R1 ¼ 0.0343, wR2 ¼ 0.0809
R1 ¼ 0.0430, wR2 ¼ 0.0858 0.06(4)
0.205 and ꢀ0.179
elaborated by Abushanabʼs group [19]. The remaining hydroxyl
group in 10 was protected as the corresponding benzyl ether 11
[20] (95%) employing BnBr and Ag2O in dry CH2Cl2. The subsequent
)

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M. Fabisíkova et al. / Carbohydrate Research 435 (2016) 26e36
29
Fig. 3. Transition state structures for the rearrangement 8/[TS1]/[TS2]/14 þ 15. Relative energies of transition states (in kcal/mol) and bond distances (in Å) are shown.
esters 12 in 77% yield over two steps (E:Z ¼ 88:12 as determined by
1H NMR spectroscopy). After their chromatographic separation, the
major ester (E)-12 [18b] (J ¼ 15.8 Hz) was then reduced with DIBAl-
H providing the required allylic alcohol 13 (93%, Scheme 2). As such,
thiocyanate 8 (87%) was cleanly produced from 13 through the
nucleophilic displacement of the corresponding in situ-generated
mesylate by KSCN. Subsequent aza-Claisen rearrangement of 8
was achieved under conventional heating as well as under
microwave-promoted thermal conditions at 70 ꢁC or 90 ꢁC in n-
heptane and afforded a separable mixture of the corresponding
isothiocyanates 14 and 15 in good combined yields (Table 1) and
with moderate selectivity (Table 1, entry 5). Prolonged heating in an
effort to increase the yield of the rearranged products (Table 1,
entry 7) was shown to be detrimental for the diastereoselective
outcome of the proceeding reaction. Table 1 (entry 7) shows that
realization of the rearrangement under thermal conditions (90 ꢁC,
17 h) exhibited an equilibrium shifted to the thermodynamically
more stable isothiocyante 15 to decrease the desired 1,2-anti-
selectivity. On the other hand, these conditions could be eventually
used to gain access to a greater amount of the 1,2-syn-diastereomer
15 as a precursor of the corresponding 2-epi-congener of spisulo-
sine 1.
representing n-heptane as the solvent.
The rearrangement of 8 occurs via transition states TS1 and TS2
with relative free energies 0 and 0.727 kcal/mol (Fig. 3). The process
is concerted but asynchronous. From the calculations, for the
pathway 8/TS1/14, the activation energy was found to be
0.727 kcal/mol lower than for the pathway 8/TS2/15. Thus, the
predicted diastereomeric ratio of 14:15 at 90 ꢁC was 73.3:26.7.
These results are in very good agreement with the experimental
data (Table 1) with the correct prediction of isothiocyanate 14 as
being the predominant diastereoisomer.
In order to rationalise the observed stereoselectivity in the [3,3]-
sigmatropic rearrangement of thiocyanate 8, high-level density
functional theory (DFT) calculations, including electron correlation
effects, were carried out. The solvent effect was taken into account
via
a
single-point calculation in
a
dielectric continuum
Fig. 4. ORTEP structure of isothiocyanate 14 showing the crystallographic numbering.
Scheme 3. Reagents and conditions: (a) (i) MeONa, MeOH, 0 ꢁC/rt, 93%; (ii) mesitylnitrile oxide (MNO), MeCN, rt; (b) (i) O3, MeOH/CH2Cl2, ‒78 ꢁC; (ii) NaBH4, ‒78 ꢁC/0 ꢁC; (c)
Ph3P, I2, imidazole, Et2O/MeCN, rt; (d) H2, 10% Pd/C, MeOH, Et3N, rt; (e) BnBr, NaH, TBAI, DMF, 0 ꢁC/rt; (f) p-TsOH, MeOH, rt; (g) (i) NaIO4, MeOH/H2O (1:1), rt; (ii) LHMDS, THF, 5, rt;
(h) H2, 10% Pd/C/20% Pd(OH)2/C (1:1), MeOH, 60 ꢁC; (j) 2 M aq NaOH, EtOH, reflux.

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M. Fabisíkova et al. / Carbohydrate Research 435 (2016) 26e36
30
Table 3
next searched for appropriate conditions for the reductive deha-
logenation. After several trials, it was found that catalytic hydro-
genation (10% Pd/C) in the presence of Et3N was the most effective
and led to the desired derivative 18 in excellent yield of 96%.
With 18 in hand, we focused on installing of the long alkyl side-
chain by the Wittig olefination. To overcome the problems with low
yields of this transformation, protection of the nitrogen atom of the
carbamate in 18 with a benzyl group was carried out to afford 19 in
92% yield (Scheme 3). Its subsequent p-toluenesulfonic acid-
mediated acetonide hydrolysis resulted in the formation of diol 6
(86%). This functionalized intermediate was then subjected to the
oxidative fragmentation with NaIO4 to produce an aldehyde, which
was then treated with a non-stabilized ylide produced from the
corresponding salt CH3(CH2)13PPh3Br 5 [17], to give a barely sepa-
rable mixture of olefins 20.
Optical rotation values for spisulosine 1 and its corresponding HCl salt 1.HCl.
Compound
Literature
Optical rotation
26
Rinehart et al. (Ref. [1b])
Chemla et al. (Ref. [11b])
[a
[a
[a
[a
[a
[a
[a
[a
[a
[a
[a
[a
[a
[a
]
]
]
]
]
]
]
]
]
]
]
]
]
]
¼ þ24.9 (c 1.0, CHCl3)
¼ þ5.2 (c 0.36, MeOH)
¼ þ24.0 (c 1.0, CHCl3)
¼ þ24.2 (c 1.0, CHCl3)
¼ þ8.2 (c 1.0, CHCl3)a
¼ þ25.3 (c 0.94, CHCl3)
¼ þ19.3 (c 0.23, CHCl3)
¼ þ8.2 (c 0.80, MeOH)
¼ þ21.4 (c 0.50, CHCl3)
¼ þ25.3 (c 0.95, CHCl3)
¼ þ16.5 (c 0.20, CHCl3)
¼ þ7.5 (c 0.30, MeOH)
¼ þ7.15 (c 0.42, MeOH)
¼ þ3.2 (c 1.0, MeOH)
D
20
D
25
D
ꢁ
Galvez et al. (Ref. [11a])
20
D
Huang et al. (Ref. [9])
Delgado et al. (Ref. [11i])
D
25
ꢁ
Padron et al. (Ref. [11j])
D
25
D
Kumar et al. (Ref. [11h])
Wang et al. (Ref. [11f])
Panda et al. (Ref. [11d])
Ghosal and Shaw (Ref. [11c])
Martinkova et al.
Sutherland et al. (Ref. [11g])
Dauban et al. (Ref. [11e])
20
D
25
D
28
D
26
ꢁ
D
23
D
24
D
25
D
ꢁ
Galvez et al. (Ref. [11a])
(88%, Z:E ¼ 94:6). Repeated chromatography of small amounts
of the obtained mixture of 20 provided only pure (Z)-20 as an
analytical sample. To complete the total synthesis, saturation of the
double bond and removal of both benzyl ether protecting groups
was achieved under catalytic hydrogenation (H2, 10% Pd/C/20%
Pd(OH)2/C, 60 ꢁC). With this procedure, compounds 20 were suc-
cessively converted into 21 (72%). Final base hydrolysis (2 M aq
NaOH, EtOH) furnished the corresponding base 1 in 82% yield
(Scheme 3). The spectroscopic data, melting point and optical
a
Temperature was not reported.
It should be noted that during this reaction we also recovered
the starting thiocyanate 8 (from 25% to 34%, Table 1) which was
repeatedly utilized in the rearrangement transformation. Purifica-
tion by column chromatography allowed the quantitative separa-
tion of both compounds 14 and 15. The major isothiocyanate 14
crystallized well from Et2O to afford suitable crystals for X-ray
diffraction analysis which unambiguously assigned the S configu-
ration of the newly formed stereocentre possessing the masked
amino functionality (Fig. 4).
rotation value for the prepared spisulosine
1 showed good
concordance with those, previously reported (for the comparison of
values of optical rotations and NMR data, see Tables 3e5,
respectively).
As we had prepared the skeletal framework with an anti-vicinal
amino-alcohol motif, our next task was to develop a suitable route
toward the final molecule 1. For this purpose, isothiocyanate 14 was
converted into carbamate 16 (87%, over two transformations) via a
two-stage process involving its reaction with MeONa followed by
mesitylnitrile oxide (MNO) treatment (Scheme 3). Subsequent
ozonolysis of 16, coupled with a reductive work up (NaBH4), pro-
vided 7 in 93% yield. To achieve the required methyl side-chain,
replacement of the free hydroxyl function in 7 with a hydrogen
atom would create the key intermediate for the synthesis of the
aforementioned 1-deoxysphingoid bases. This deoxygenation was
accomplished in a two-step approach. First, the alcohol 7 was
transformed to iodide 17 in 85% yield according to the protocol
reported by Gareggʼs group [22]. Having obtained compound 17, we
2.2. Antiproliferative/cytotoxic activity
Four newly prepared compounds along with the commercially
available anticancer substance cisplatin were evaluated for their
in vitro antiproliferative activity on a panel of 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
leukaemia), HeLa (cervical adenocarcinoma), and a non-malignant
cell line NiH 3T3 (mouse fibroblasts) using the MTT assay. The re-
sults are summarized in Table 6 as IC50 values. It should be noted
that cytotoxicities shown here are lower than those published for 1
on the other cancer cells (P-388, A-549, HT-29, MeL-28, DU-145) by
Acena and co-workers [5b]. However, the lack of more detailed
Table 4
1H NMR data for spisulosine 1 and its corresponding HCl salt 1.HCl.
Compound
Literature
Solvent
H-1 (CH3)
H-2
H-3
H-18 (CH3)
Chemla et al. (Ref. [11b])
CD3OD
CD3OD
CD3OD
CD3OD
CDCl3
CD3OD
CDCl3
CD3OD
CDCl3
1.24 d
1.03.d
1.05 d
1.04 d
1.05 d
1.17 d
1.00 d
1.20 d
0.99 d
1.01 d
1.05 d
1.03 d
1.12 d
1.21 d
1.17 d
3.29 dq
2.77 qd
2.75e2.87 m
2.78 m
3.02 br s
3.21e3.24 m
2.97 m
3.22e3.34 m
2.94 br m
2.88e3.06 m
2.81 qd
2.76e2.82 m
3.14e3.20 m
3.27 qd
3.72 m
3.40 ddd
3.44 m
0.93 t
0.89 t
0.91 t
0.90 t
0.88 t
0.83e0.87 m
0.88 t
0.89 t
0.84e0.88 m
0.88 t
ꢁ
Galvez et al. (Ref. [11a])
Huang et al. (Ref. [9])
Delgado et al. (Ref. [11i])
3.40 m
ꢁ
Padron et al. (Ref. [11j])
3.52 br s
3.65e3.71 m
3.44 m
Kumar et al.(Ref. [11h])
Wang et al. (Ref. [11f])
Panda et al.(Ref. [11d])
Ghosal and Shaw (Ref. [11c])
Coelho et al. (Ref. [13])a
Reinhart et al.(Ref. [1b])
3.68 m
3.42 br m
3.39e3.47 m
3.42 dt
3.38e3.42 m
3.57e3.62 m
3.69 td
CDCl3
CD3OD
CD3OD
CD3OD
CD3OD
CD3OD
0.89 t
0.87 t
0.80 t
0.90 t
ꢁ
Martinkova et al.
Sutherland et al. (Ref. [11g])
Dauban et al. (Ref. [11e])
ꢁ
Galvez et al. (Ref. [11a])
3.23 qd
3.62e3.70 m
0.86 t
a
Data for ( )-spisulosine.

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M. Fabisíkova et al. / Carbohydrate Research 435 (2016) 26e36
31
Table 5
13C NMR data for spisulosine 1 and its corresponding HCl salt 1.HCl.
Compound
Literature
Solvent
C-1
C-2
C-3
C-18
Chemla et al. (Ref. [11b])
CD3OD
CD3OD
CD3OD
CD3OD
CDCl3
CDCl3
CDCl3
CDCl3
CDCl3
15.3
17.3
17.0
17.2
16.0
17.0
17.0
17.1
17.3
16.8
16.8
16.8
14.5
14.4
14.5
53.5
52.2
52.3
52.1
50.6
51.6
NRa
50.5
50.8
50.3
52.3
52.2
52.6
52.6
52.7
72.6
76.6
76.3
76.5
74.0
70.6
NRa
74.9
75.1
74.7
76.2
76.1
71.7
71.7
71.7
13.1
14.5
14.5
14.5
14.1
13.4
14.3
14.3
14.5
14.1
14.6
14.5
12.1
12.1
12.1
ꢁ
Galvez et al. (Ref. [11a])
Huang et al. (Ref. [9])
Delgado et al. (Ref. [11i])
ꢁ
Padron et al. (Ref. [11j])
Kumar et al. (Ref. [11h])
Wang et al. (Ref. [11f])
Panda et al. (Ref. [11d])
Ghosal and Shaw (Ref. [11c])
Coelho et al. (Ref. [13])b
Reinhart et al. (Ref. [1b])
CDCl3
CD3OD
CD3OD
CD3OD
CD3OD
CD3OD
ꢁ
Martinkova et al.
Sutherland et al. (Ref. [11g])
Dauban et al. (Ref. [11e])
ꢁ
Galvez et al. (Ref. [11a])
a
Not reported.
Data for ( )-spisulosine.
b
Table 6
Antiproliferative activities on six human cancer cell lines (MDA-MB-231, MCF-7, HCT-116, Caco-2, Jurkat, HeLa) and non-malignant mouse fibroblasts NiH 3T3.
a
Compd no.
Cell line, IC50
MDA-MB-231
SD (
m
mol ꢂ Lꢀ1
)
MCF-7
HCT-116
Caco-2
Jurkat
NiH 3T3
HeLa
1
14
17
21
3.45 1.08
31.4 1.7
>100
>100
17.5
ꢃ1
<1
<1
ꢃ1
3.25 0.68
29.1 0.14
>100
>100
20.87
ꢃ1
33.03 7.13
>100
<10
>100
>100
20.3
30.4 13.44
>100
>100
23
5
<10
>100
>100
14.1
>100
>100
16.2
>100
15.6
cisplatin
17.2
a
The potency of the listed compounds was determined using the MTT assay after 72 h incubation of cells and given as IC50 (concentration of a tested compound that
decreased the amount of viable cells to 50% relative to untreated control cells, see Section 4.2).
experimental data, including incubation time, given by the afore-
mentioned research group has concluded further comparison.
The antiproliferative activity results (Table 6) revealed that
spisulosine exhibited significant potency, mainly against MCF-7,
HeLa, CaCo-2, HCT-116 and Jurkat cells. On the other hand, the
protected form of spisulosine, carbamate 21, was completely inac-
tive in all tested cells suggesting a key role of the free vicinal amino
alcohol moiety. The major isothiocyanate 14 demonstrated com-
parable or higher in vitro activity than the commercially available
anticancer drug cisplatin on HCT-116 and HeLa cell lines, respec-
tively. These finding observed from the comparison of the cytotoxic
activity of compounds 14 and 21 could be explained either via the
different mode of action of 14 due to presumably by the presence of
the NCS group or by its diverse targets in the tested cancer lines.
non-malignant mouse fibroblasts (NiH 3T3). These findings support
our further screening of 1 against 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, 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: hexane with
CaCl2, ethyl acetate with K2CO3, methanol with BaO and CH2Cl2
with CaH2. Thin layer chromatography was run on Merck silica gel
60 F254 analytical plates; detection was carried out with either ul-
traviolet light (254 nm), or spraying with a solution of phospho-
molybdic acid, a basic potassium permanganate solution, or a
solution of concentrated 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) spectrometer using TMS as the internal refer-
3. Conclusions
In summary, we have reported the synthesis of spisulosine 1.
The described approach relied on the substrate-controlled aza-
Claisen rearrangement which established the desired amino-
alcohol unit and a subsequent deoxygenation step which installed
the methyl side framework. A Wittig olefination step permitted
coupling of the polar fragment 6 with the hydrophobic chain 5 to
create the final carbon backbone of 1. Spisulosine 1 was assessed for
its antiproliferative activity against six cancer cell lines (Jurkat,
HeLa, MDA-MB-231, MCF-7, HTC-116 and Caco-2) and displayed
significant potency with IC50 values down to sub-micromolar
range. In addition, substance 1 demonstrates quite promising
selectivity in cytotoxicity discriminating between the tested cancer
cell lines (MCF-7, CaCo-2, HCT-116, Jurkat and HeLa) and
ence. For 1H,
¼ 0.0), CD3OD (
CDCl3
¼ 77.00), CD3OD (
d are given in parts per million (ppm) relative to TMS
(d
d
¼ 4.84), C6D6
(
d
¼ 7.15) and for 13C relative to
(d
d
¼ 49.05), C6D6
(d
¼ 128.02). For the
assignment of resonances, homonuclear COSY and heteronuclear
2D correlated (HSQC, HMBC) techniques were used. Infrared (IR)
spectra were measured with a Nicolet 6700 FT-IR spectrometer and

ꢀ
ꢁ
M. Fabisíkova et al. / Carbohydrate Research 435 (2016) 26e36
32
expressed in v values (cmꢀ1). Optical rotations were measured on a
P-2000 Jasco polarimeter and reported as follows: [ D (c in grams
(2 ꢂ CHPh), 137.9 (Ci). Anal. Calcd for C14H20O4: C, 66.65; H, 7.99.
a]
Found: C, 66.72; H, 7.93.
per 100 mL, solvent). Melting points were recorded on a Kofler hot
block, and are uncorrected. Microwave reactions were carried out
on the focused microwave system (CEM Discover). The temperature
content of the vessel was monitored using a calibrated infrared
sensor mounted under the vessel. At the end of all reactions the
contents of the vessel were cooled rapidly using a stream of com-
pressed air. Small quantities of reagents (mL) were measured with
appropriate syringes (Hamilton). All reactions were performed
under an atmosphere of nitrogen, unless otherwise noted.
4.1.4. Ethyl (4S,2E)-4-(benzyloxy)-4-[(40R)-20,2'-dimethyl-10,3'-
dioxolan-4'-yl]but-2-enoate (E-12) and ethyl (4S,2Z)-4-
(benzyloxy)-4-[(40R)-20,2'-dimethyl-10,3'-dioxolan-4'-yl]but-2-
enoate (Z-12)
To a solution of 9 (2.82 g, 11.2 mmol) in MeCN (85 mL) was
added IBX (6.27 g, 22.4 mmol) and the resulting mixture was stirred
at reflux for 1 h. After cooling to room temperature, the insoluble
parts were filtered off, the solvent was removed in vacuo, and the
residue was subjected to the next reaction without further
purification.
4.1.1. Ethyl (R)-2-[(40R)-20,2'-dimethyl-1,3-dioxolan-4'-yl]-2-
hydroxyacetate (10) [19]
For the preparation of 10 we used the known procedure re-
ported in Ref. [20]. Here we would like to present the full spectral
A solution of the crude aldehyde (2.80 g, 11.2 mmol) in dry
CH2Cl2 (95 mL) was treated with the stabilized ylide
(Ph3P ¼ CHCO2Et, 4.67 g, 13.4 mmol) and stirring was continued for
1 h at room temperature. After completion of the reaction, the
solvent was evaporated, and the residue was chromatographed on
silica gel (n-hexane/ethyl acetate, 11:1) to afford 2.76 g (77%) of a
mixture of esters 12 [18b]. Only (E)-12 was isolated in the pure form
data of 10 along with its value of the optical rotation; [
0.72, MeOH); lit [19]. [
reported). IR ymax 3447, 2985, 2937, 2904, 1733, 1371, 1250, 1206,
a
]
25 ꢀ 24.3 (c
D
a]
D ꢀ 29.14 (c 1.565, MeOH, temperature not
1065 cmꢀ1; 1H NMR (400 MHz, CDCl3):
d
1.32 (t, 3H, J ¼ 7.1 Hz, CH3),
1.36 (s, 3H, CH3), 1.44 (s, 3H, CH3), 2.89 (d, 1H, J ¼ 6.3 Hz, OH),
4.00e4.06 (m, 2H, 2 ꢂ H-50), 4.24e4.35 (m, 4H, H-2, CH2, H-40); 13C
as a colourless oil; [
a
]
27 þ 33.5 (c 0.53, CHCl3); lit [18b]. [
a
]
25 þ 11.0
D
D
NMR (100 MHz, CDCl3):
d 14.1 (CH3), 25.1 (CH3), 26.3 (CH3), 61.9
(c 1.3, CHCl3). IR ymax 2984, 2936, 2874, 1717, 1658, 1455, 1369, 1264,
(CH2), 65.0 (CH2, C-50), 71.1 (CH, C-2), 77.0 (CH, C-40), 109.9 (Cq),
172.0 (C]O). Anal. Calcd for C9H16O5: C, 52.93; H, 7.90. Found: C,
52.85; H, 7.98.
1210, 1174, 1070 cmꢀ1 1H NMR (400 MHz, CDCl3):
; d 1.31 (t, 3H,
J ¼ 7.2 Hz, CH3), 1.33 (s, 3H, CH3), 1.41 (s, 3H, CH3), 3.88 (dd, 1H,
J ¼ 8.4 Hz, J ¼ 5.2 Hz, H-50), 3.96 (dt, 1H, J ¼ 6.4 Hz, J ¼ 6.4 Hz,
J ¼ 1.0 Hz, H-4), 4.05 (dd, 1H, J ¼ 8.4 Hz, J ¼ 6.4 Hz, H-50), 4.09e4.13
(m, 1H, H-40), 4.22 (q, 2H, J ¼ 7.1 Hz, CH2), 4.43 (d, 1H, J ¼ 11.7 Hz,
OCH2Ph), 4.65 (d, 1H, J ¼ 11.7 Hz, OCH2Ph), 6.10 (dd, 1H, J ¼ 15.8 Hz,
J ¼ 0.9 Hz, H-2), 6.92 (dd, 1H, J ¼ 15.8 Hz, J ¼ 6.2 Hz, H-3), 7.21e7.44
4.1.2. Ethyl (R)-2-(benzyloxy)-2-[(40R)-20,2'-dimethyl-10,3'-
dioxolan-4'-yl]acetate (11) [20]
Applying the same reaction conditions described in Ref. [20a],
compound 10 [19] (0.50 g, 2.40 mmol), benzyl bromide (0.30 mL,
2.60 mmol) and Ag2O (0.85 g, 3.60 mmol) in dry CH2Cl2 (5 mL)
afforded after stirring (1.5 h), treatment and chromatography on
(m, 5H, Ph); 13C NMR (100 MHz, CDCl3):
d 14.2 (CH3), 25.2 (CH3),
26.6 (CH3), 60.6 (CH2), 66.6 (CH2, C-50), 71.7 (OCH2Ph), 77.2 (CH, C-
40), 78.8 (CH, C-4), 109.8 (Cq), 124.0 (CH, C-2), 127.8 (2 ꢂ CHPh), 127.9
(CHPh), 128.4 (2 ꢂ CHPh), 137.4 (Ci), 144.6 (CH, C-3), 165.8 (C]O).
Anal. Calcd for C18H24O5: C, 67.48; H, 7.55. Found: C, 67.54; H, 7.49.
silica gel (n-hexane/ethyl acetate, 9:1) 0.68 g (95%) of compound 11
25
as a colourless viscous oil; [
a
]
þ 43.5 (c 0.41, CHCl3); lit [20a].
D
[
a]
20 þ 26.5 (c 2.0, MeOH); lit [21]. [
a
]
20 ꢀ 46.0 (c 2.0, CHCl3) for ent-
D
D
11. IR ymax 2985, 2936, 2875, 1741, 1455, 1371, 1074 cmꢀ1
;
1H NMR
(400 MHz, CDCl3):
d
1.29 (t, 3H, J ¼ 7.1 Hz, CH3), 1.33 (s, 3H, CH3),
1.42 (s, 3H, CH3), 3.95 (d, 1H, J ¼ 6.4 Hz, H-2), 3.98e4.05 (m, 2H,
2 ꢂ H-50), 4.18e4.28 (m, 2H, CH2), 4.31e4.36 (m, 1H, H-40), 4.50 (d,
1H, J ¼ 11.6 Hz, OCH2Ph), 4.68 (d, 1H, J ¼ 11.6 Hz, OCH2Ph),
4.1.5. 4.1.5. (4S,2E)-4-(Benzyloxy)-4-[(40R)-20,2'-dimethyl-10,3'-
dioxolan-4'-yl]but-2-en-1-ol (13)
To a solution of (E)-12 (2.43 g, 7.58 mmol) in dry CH2Cl2 (34 mL)
that had been pre-cooled to ꢀ50 ꢁC was added dropwise DIBAl-H
(18.9 mL, 22.7 mmol, ~1.2 M solution in toluene) for 15 min, and
the resulting solution was stirred at ꢀ50 ꢁC for another 30 min. The
excess hydride was decomposed by the cautious addition of MeOH
(4.4 mL). Then, a 30% solution of K/Na tartrate (114 mL) was added
and stirring was continued at room temperature for 2 h. After
separation of the organic layer, the aqueous phase was extracted
with CH2Cl2 (2 ꢂ 150 mL). The combined organic extracts were
dried over Na2SO4, the solvent was removed, and the residue was
subjected to flash chromatography on silica gel (n-hexane/ethyl
7.28e7.37 (m, 5H, Ph); 13C NMR (100 MHz, CDCl3):
d 14.2 (CH3), 25.3
(CH3), 26.6 (CH3), 61.1 (CH2), 66.2 (CH2, C-50), 72.8 (OCH2Ph), 76.0
(CH, C-40), 79.1 (CH, C-2), 109.8 (Cq), 128.0 (CHPh), 128.1 (2 ꢂ CHPh),
128.4 (2 ꢂ CHPh), 137.0 (Ci), 170.4 (C]O). Anal. Calcd for C16H22O5:
C, 65.29; H, 7.53. Found: C, 65.23; H, 7.61.
4.1.3. (2S)-2-(Benzyloxy)-2-[(40R)-20,2'-dimethyl-10,3'-dioxolan-4'-
yl]ethanol (9) [18]
Using the same reaction conditions as described in Ref. [18b],
compound 11 (0.68 g, 2.30 mmol) and LiAlH4 (0.175 g, 4.61 mmol)
in THF (9 mL) afforded after stirring at ambient temperature (1 h),
treatment and chromatography through a short column of silica gel
(n-hexane/ethyl acetate, 3:1) 0.52 g (89%) of 9 as a colourless oil;
acetate, 2:1). This procedure yielded 1.96 g (93%) of compound 13
27
as a colourless oil; [
a]
þ 53.3 (c 0.78, CHCl3). IR ymax 3417, 2985,
D
2869, 1454, 1371, 1253, 1209, 1155, 1046 cmꢀ1; 1H NMR (400 MHz,
CDCl3): 1.34 (s, 3H, CH3), 1.41 (s, 3H, CH3), 2.02 (br s, 1H, OH), 3.78
26
27
[
a]
þ 22.1 (c 0.67, CHCl3); lit [18a]. [
a
]
þ 21.7 (c 1.0, CHCl3); lit
d
D
D
25
[18b]. [
a]
þ 23.3 (c 1.1, CHCl3). IR ymax 3446, 2985, 2919, 1454,
(app. t, 1H, J ¼ 6.9 Hz, H-4), 3.87 (dd, 1H, J ¼ 8.1 Hz, J ¼ 5.3 Hz, H-50),
4.03e4.13 (m, 2H, H-40, H-50), 4.18e4.22 (m, 2H, 2 ꢂ H-1), 4.41 (d,
1H, J ¼ 11.7 Hz, OCH2Ph), 4.63 (d, 1H, J ¼ 11.7 Hz, OCH2Ph), 5.69 (dd,
1H, J ¼ 15.7 Hz, J ¼ 7.7 Hz, H-3), 5.92 (app. td, 1H, J ¼ 15.6 Hz,
J ¼ 5.2 Hz, H-2), 7.25e7.36 (m, 5H, Ph); 13C NMR (100 MHz, CDCl3):
D
1370, 1210, 1068 cmꢀ1
;
1H NMR (400 MHz, CDCl3):
d 1.35 (s, 3H,
CH3), 1.42 (s, 3H, CH3), 2.18 (br s, 1H, OH), 3.52 (app. td, 1H,
J ¼ 6.7 Hz, J ¼ 4.2 Hz, H-2), 3.65e3.74 (m, 1H, H-1), 3.83 (dd, 1H,
J ¼ 11.9 Hz, J ¼ 3.9 Hz, H-1), 3.87 (dd, 1H, J ¼ 8.4 Hz, J ¼ 5.9 Hz, H-50),
4.08 (dd, 1H, J ¼ 8.4 Hz, J ¼ 6.4 Hz, H-50), 4.15e4.22 (m, 1H, H-40),
4.63 (d, 1H, J ¼ 11.6 Hz, OCH2Ph), 4.67 (d, 1H, J ¼ 11.6 Hz, OCH2Ph)
d
25.2 (CH3), 26.5 (CH3), 62.7 (CH2, C-1), 66.8 (CH2, C-50), 70.6
(OCH2Ph), 77.6 (CH, C-40), 80.0 (CH, C-4), 109.5 (Cq), 127.6 (CHPh),
127.8 (2 ꢂ CHPh), 128.0 (CH, C-3), 128.3 (2 ꢂ CHPh), 134.5 (CH, C-2),
138.1 (Ci). Anal. Calcd for C16H22O4: C, 69.04; H, 7.97. Found: C,
69.11; H, 7.89.
7.25e7.40 (m, 5H, Ph); 13C NMR (100 MHz, CDCl3):
d 25.2 (CH3), 26.6
(CH3), 61.7 (CH2, C-1), 66.9 (CH2, C-50), 72.6 (OCH2Ph), 75.8 (CH, C-
40), 79.6 (CH, C-2), 109.2 (Cq), 127.9 (2 ꢂ CHPh), 128.0 (CHPh), 128.5

ꢀ
ꢁ
M. Fabisíkova et al. / Carbohydrate Research 435 (2016) 26e36
33
4.1.6. 4.1.6. (4R)-4-[(10S,20E)-1'-(Benzyloxy)-4'-thiocyanatobut-2'-
en-1'-yl]-2,2-dimethyl-1,3-dioxolane (8)
7.22e7.40 (m, 5H, Ph); 13C NMR (100 MHz, CDCl3):
d
25.2 (CH3), 26.7
(CH3), 61.5 (CH, C-20), 66.6 (CH2, C-5), 74.4 (OCH2Ph), 74.8 (CH, C-4),
81.6 (CH, C-10), 109.4 (Cq), 119.3 (CH2, C-40), 128.0 (2 ꢂ CHPh), 128.1
(CHPh), 128.5 (2 ꢂ CHPh), 131.2 (CH, C-30), 134.8 (C, NCS), 137.3 (Ci).
Anal. Calcd for C17H21NO3S: C, 63.92; H, 6.63; N, 4.39. Found: C,
To a solution of 13 (1.91 g, 6.86 mmol) in dry CH2Cl2 (59 mL) that
had been pre-cooled to 0 ꢁC were successively added Et3N (1.5 mL,
10.3 mmol) and MsCl (0.80 mL, 10.3 mmol), and stirring was
continued for 30 min at 0 ꢁC. Then, the solvent was evaporated and
the residue was diluted with Et2O (65 mL) to produce the insoluble
salt, which was removed by filtration. The filtrate was concentrated
in vacuo and the obtained product was subjected to the next re-
action without further purification.
A solution of the crude mesylate (2.44 g, 6.86 mmol) in dry
MeCN (49 mL) was treated with KSCN (1.27 g, 13.0 mmol) at 0 ꢁC.
After stirring for 15 min at 0 ꢁC and then at room temperature for
another 20 h, the solvent was evaporated, the residue was diluted
with Et2O (45 mL), and the solid part was filtered off. Evaporation of
the solvent and chromatography of the residue on silica gel (n-
63.99; H, 6.58; N, 4.34.
25
Diastereomer 15: colourless viscous oil; [
a
]
þ 125.2 (c 0.54,
D
CHCl3). IR ymax 3031, 2986, 2930, 2884, 2079, 2052,1645,1454,1371,
1254, 1210, 1070 cmꢀ1 1H NMR (400 MHz, CDCl3):
;
d
1.34 (s, 3H,
CH3), 1.40 (s, 3H, CH3), 3.57 (dd, 1H, J ¼ 7.2 Hz, J ¼ 2.5 Hz, H-10), 3.82
(dd, 1H, J ¼ 8.5 Hz, J ¼ 5.4 Hz, H-5), 4.04 (dd, 1H, J ¼ 8.5 Hz,
J ¼ 6.3 Hz, H-5), 4.13e4.19 (m, 1H, H-4), 4.48e4.51 (m, 1H, H-20),
4.60 (d, 1H, J ¼ 11.4 Hz, OCH2Ph), 4.74 (d, 1H, J ¼ 11.4 Hz, OCH2Ph),
5.32 (dd, 1H, J ¼ 10.3 Hz, J ¼ 1.1 Hz, H-40), 5.48 (dd, 1H, J ¼ 16.8 Hz,
J ¼ 1.3 Hz, H-40), 5.93 (ddd, 1H, J ¼ 16.8 Hz, J ¼ 10.3 Hz, J ¼ 5.5 Hz, H-
30), 7.24e7.39 (m, 5H, Ph); 13C NMR (100 MHz, CDCl3):
d 26.1 (CH3),
hexane/ethyl acetate, 7:1) gave 1.91 g (87%) of compound 8 as a
26.7 (CH3), 61.2 (CH, C-20), 66.5 (CH2, C-5), 74.7 (OCH2Ph), 75.1 (CH,
C-4), 81.5 (CH, C-10), 109.4 (Cq), 117.8 (CH2, C-40), 127.7 (2 ꢂ CHPh),
128.0 (CHPh), 128.4 (2 ꢂ CHPh), 133.2 (CH, C-30), 135.3 (C, NCS), 137.3
(Ci). Anal. Calcd for C17H21NO3S: C, 63.92; H, 6.63; N, 4.39. Found: C,
63.86; H, 6.68; N, 4.44.
27
light yellow oil; [
a
]
þ 35.9 (c 0.72, CHCl3). IR ymax 2985, 2934,
D
2873, 2154, 1496, 1454, 1370, 1209, 1070 cmꢀ1; 1H NMR (400 MHz,
CDCl3):
d
1.34 (s, 3H, CH3), 1.41 (s, 3H, CH3), 3.59 (dd, 1H, J ¼ 13.0 Hz,
J ¼ 5.7 Hz, H-40), 3.64 (dd, 1H, J ¼ 13.0 Hz, J ¼ 6.6 Hz, H-40),
3.80e3.85 (m, 1H, H-10), 3.85e3.92 (m, 1H, H-5), 4.05e4.12 (m, 2H,
H-4, H-5), 4.47 (d, 1H, J ¼ 11.7 Hz, OCH2Ph), 4.70 (d, 1H, J ¼ 11.7 Hz,
OCH2Ph), 5.80e5.92 (m, 2H, H-20, H-30), 7.24e7.38 (m, 5H, Ph); 13C
4.1.8. 4.1.8. Methyl {(1S,2S)-1-(benzyloxy)-1-[(40R)-20,2'-dimethyl-
10,3'-dioxolan-4'-yl]but-3-en-2-yl}carbamate (16)
NMR (100 MHz, CDCl3):
d
25.2 (CH3), 26.6 (CH3), 35.7 (CH2, C-40),
Sodium methoxide (98 mg, 1.82 mmol) was added to a solution
of 14 (0.45 g, 1.4 mmol) in dry MeOH (14 mL) that had been pre-
cooled to 0 ꢁC. After stirring for 30 min at 0 ꢁC and then at room
temperature for another 7 h, the solvent was evaporated, and the
residue was partitioned between CH2Cl2 (30 mL) and water (15 mL).
The organic layer was separated and the aqueous one was washed
with another portion of CH2Cl2 (30 mL). The combined organic
extracts were dried over Na2SO4 and concentrated. The crude
product was subjected to flash chromatography on silica gel (n-
66.8 (CH2, C-5), 71.0 (OCH2Ph), 77.5 (CH, C-4), 79.2 (CH, C-10), 109.6
(Cq), 111.6 (C, SCN), 126.9 (CH, C-30), 127.8 (CHPh), 128.0 (2 ꢂ CHPh),
128.4 (2 ꢂ CHPh), 134.6 (CH, C-20), 137.7 (Ci). Anal. Calcd for
C
17H21NO3S: C, 63.92; H, 6.63; N, 4.39. Found: C, 63.99; H, 6.57; N,
4.44.
4.1.7. 4.1.7. (4R)-4-[(10S,20S)-1'-(Benzyloxy)-2'-isothiocyanatobut-
3'-en-1'-yl]-2,2-dimethyl-1,3-dioxolane (14) and (4R)-4-[(10S,20R)-
1'-(benzyloxy)-2'-isothiocyanatobut-3'-en-1'-yl]-2,2-dimethyl-1,3-
dioxolane (15)
4.1.7.1. Conventional method. A solution of 8 (0.10 g, 0.31 mmol) in
n-heptane (2.6 mL) was stirred and heated under a nitrogen at-
mosphere (for the temperatures and reaction times, see Table 1).
After completion of the reaction (judged by TLC), the mixture was
cooled to room temperature and concentrated. The residue was
purified by flash chromatography through a short column of silica
gel (n-hexane/ethyl acetate, 11:1) to afford the corresponding iso-
thiocyanates 14 and 15 (for the combined yields, see Table 1).
hexane/ethyl acetate, 9:1) to give 0.46 g (93%) of thiocarbamate as a
24
colourless oil; [
a]
ꢀ 39.7 (c 0.37, CHCl3), which was used in the
D
next transformation without spectral characterization due to the
presence of rotamers.
A solution of the obtained thiocarbamate (0.45 g, 1.28 mmol) in
dry MeCN (12.6 mL) was treated with MNO (0.27 g, 1.66 mmol) and
the resulting mixture was stirred at room temperature for 1 h.
Evaporation of the solvent and chromatography of the residue on
silica gel (n-hexane/ethyl acetate, 5:1) gave 0.40 g (93%) of com-
23
pound 16 as a colourless oil; [
a]
ꢀ 18.5 (c 0.86, CHCl3). IR ymax
D
3330, 2985, 2935, 1702, 1643, 1510, 1454, 1370, 1218, 1067 cmꢀ1; 1H
NMR (400 MHz, CDCl3): 1.33 (s, 3H, CH3), 1.43 (s, 3H, CH3), 3.65 (s,
4.1.7.2. Microwave-assisted synthesis. A starting thiocyanate
8
d
(0.1 g, 0.31 mmol) was weight into a 10-mL glass pressure micro-
wave tube equipped with a magnetic stirbar and dissolved in n-
heptane (2.6 mL). The tube was closed with a silicon septum and
the mixture was subjected to microwave irradiation (for the tem-
peratures and reaction times, see Table 1). After cooling to room
temperature, the solvent was evaporated, and the residue was
chromatographed on silica gel (n-hexane/ethyl acetate,11:1) to give
isothiocyanates 14 and 15 (for the combined yields, see Table 1).
To require a greater amount of the rearranged products, they
were prepared at 90 ꢁC under the conventional heating.
3H, OCH3), 3.70e3.74 (m, 1H, H-1), 3.88 (dd, 1H, J ¼ 8.2 Hz,
J ¼ 6.3 Hz, H-50), 4.01 (dd, 1H, J ¼ 8.2 Hz, J ¼ 6.3 Hz, H-50), 4.10 (m,
1H, H-40), 4.39e4.47 (m, 1H, H-2), 4.62 (d, 1H, J ¼ 11.5 Hz, OCH2Ph),
4.73 (d, 1H, J ¼ 11.5 Hz, OCH2Ph), 4.96 (br d, 1H, J ¼ 6.2 Hz, NH), 5.20
(d, 1H, J ¼ 10.4 Hz, H-4), 5.27 (d, 1H, J ¼ 17.4 Hz, H-4), 5.84 (ddd, 1H,
J ¼ 17.4 Hz, J ¼ 10.4 Hz, J ¼ 7.2 Hz, H-3), 7.24e7.40 (m, 5H, Ph); 13
C
NMR (100 MHz, CDCl3):
d 25.1 (CH3), 26.6 (CH3), 52.1 (OCH3), 55.2
(CH, C-2), 66.3 (CH2, C-5ˈ), 74.3 (OCH2Ph), 76.2 (CH, C-40), 81.5 (CH,
C-1), 108.8 (Cq), 117.6 (CH2, C-4), 127.8 (CHPh), 127.9 (CHPh), 128.5
(3 ꢂ CHPh), 134.1 (CH, C-3), 137.9 (Ci), 156.2 (C]O). Anal. Calcd for
Diastereomer 14: white crystals; mp 53e54 ꢁC (recrystallized
C18H25NO5: C, 64.46; H, 7.51; N, 4.18. Found: C, 64.39; H, 7.56; N,
26
from Et2O); [
a
]
ꢀ 37.8 (c 0.64, CHCl3). IR ymax 2978, 2927, 2885,
4.23.
D
2183, 2132, 1648, 1454, 1412, 1380, 1370, 1354, 1262, 1207, 1154,
1103, 1065, 1044, 1009 cmꢀ1 1H NMR (400 MHz, CDCl3):
;
d
1.32 (s,
4.1.9. 4.1.9. Methyl {(1S,2S)-1-(benzyloxy)-1-[(40R)-20,2'-dimethyl-
10,3'-dioxolan-4'-yl]-3-hydroxypropan-2-yl}carbamate (7)
Ozone was introduced to a solution of 16 (0.39 g, 1.2 mmol) in
EtOH (12.6 mL) that had been pre-cooled to ꢀ78 ꢁC for 10 min. After
the complete consumption of the starting material, nitrogen was
passed through the cold solution for 5 min in order to remove the
excess ozone. The resulting mixture was treated with NaBH4
3H, CH3), 1.41 (s, 3H, CH3), 3.69 (dd, 1H, J ¼ 6.9 Hz, J ¼ 2.8 Hz, H-10),
3.81e3.89 (m, 1H, H-5), 3.99e4.05 (m, 2H, H-4, H-5), 4.52 (tdd, 1H,
J ¼ 6.6 Hz, J ¼ 2.6 Hz, J ¼ 1.2 Hz, J ¼ 1.2 Hz, H-20), 4.66 (d, 1H,
J ¼ 11.2 Hz, OCH2Ph), 4.83 (d, 1H, J ¼ 11.2 Hz, OCH2Ph), 5.35 (dd, 1H,
J ¼ 10.2 Hz, J ¼ 0.7 Hz, H-40), 5.42 (dd, 1H, J ¼ 16.9 Hz, J ¼ 0.7 Hz, H-
40), 5.92 (ddd, 1H, J ¼ 16.9 Hz, J ¼ 10.2 Hz, J ¼ 6.6 Hz, H-30),

ꢀ
ꢁ
M. Fabisíkova et al. / Carbohydrate Research 435 (2016) 26e36
34
(0.20 g, 5.4 mmol), and stirring was continued for 30 min at ꢀ78 ꢁC
and then at 0 ꢁC for further 30 min. The reaction was quenched by
neutralization with a 1 M HCl solution and the solvent was evap-
orated. The residue was diluted with CH2Cl2 (15 mL) and gradually
washed with H2O (10 mL) and a saturated NaCl solution (10 mL).
The organic layer was dried over Na2SO4, the solvent was evapo-
rated, and the residue was flash-chromatographed on silica gel (n-
d 15.6 (CH3), 25.2 (CH3), 26.6 (CH3), 48.1 (CH, C-2), 52.0 (OCH3), 66.8
(CH2, C-50), 74.4 (OCH2Ph), 76.1 (CH, C-40), 81.6 (CH, C-1), 109.0 (Cq),
127.8 (CHPh), 127.9 (CHPh), 128.5 (3 ꢂ CHPh), 138.1 (Ci), 156.2 (C]O).
Anal. Calcd for C17H25NO5: C, 63.14; H, 7.79; N, 4.33. Found: C,
63.20; H, 7.74; N, 4.37.
4.1.12. 4.1.12. Methyl benzyl{(1S,2S)-1-(benzyloxy)-1-[(40R)-20,2'-
dimethyl-10,3'-dioxolan-4'-yl]propan-2-yl}carbamate (19)
To a solution of 18 (0.16 g, 0.49 mmol) in dry DMF (1.8 mL) that
had been pre-cooled to 0 ꢁC were successively added NaH (23.5 mg,
0.98 mmol, ~60% suspension in mineral oil), BnBr (0.12 mL,
hexane/ethyl acetate, 1:1). This procedure yielded 0.37 g (93%) of
24
alcohol 7 as a colourless oil; [
a
]
ꢀ 1.47 (c 0.62, CHCl3). IR ymax
D
3329, 2985, 2936, 1698, 1521, 1455, 1371, 1212, 1060 cmꢀ1; 1H NMR
(400 MHz, CDCl3): 1.35 (s, 3H, CH3), 1.44 (s, 3H, CH3), 3.63e3.68
d
(m, 4H, OCH3, H-3), 3.70 (dd, 1H, J ¼ 6.1 Hz, J ¼ 4.6 Hz, H-1),
3.87e3.93 (m, 2H, H-3, H-50), 3.93e3.96 (m, 1H, H-2), 4.08e4.14 (m,
1H, H-50), 4.16e4.22 (m, 1H, H-40), 4.65 (d, 1H, J ¼ 11.3 Hz, OCH2Ph),
4.70 (d, 1H, J ¼ 11.3 Hz, OCH2Ph), 5.41 (d, 1H, J ¼ 8.3 Hz NH),
0.98 mmol) and TBAI (3.7 mg, 9.8
0
mmol). After stirring for 15 min at
ꢁC and then at room temperature for further 45 min, MeOH
(0.1 mL) was added to decompose the excess hydride followed by
addition of the cold water (18 mL). The mixture was extracted with
Et2O (2 ꢂ 20 mL), the combined organic extracts were dried over
Na2SO4, and the solvent was evaporated in vacuo. Chromatography
of the residue through a short column of silica gel (n-hexane/ethyl
7.26e7.39 (m, 5H, Ph); 13C NMR (100 MHz, CDCl3):
d 25.2 (CH3), 26.4
(CH3), 52.2 (OCH3), 52.3 (CH, C-2), 62.1 (CH2, C-3), 66.8 (CH2, C-50),
74.0 (OCH2Ph), 75.3 (CH, C-40), 80.5 (CH, C-1), 109.5 (Cq), 128.1
(3 ꢂ CHPh), 128.6 (2 ꢂ CHPh), 137.4 (Ci), 156.9 (C]O). Anal. Calcd for
acetate, 5:1) gave 0.19 g (92%) of compound 19 as a colourless oil;
27
C
17H25NO6: C, 60.16; H, 7.42; N, 4.13. Found: C, 60.23; H, 7.36; N,
[a
]
ꢀ 22.0 (c 0.20, CHCl3). IR ymax 2985, 2935, 1696, 1451, 1370,
D
4.17.
1209, 1059 cmꢀ1 1H NMR (400 MHz, CDCl3, 60 ꢁC):
; d 1.17 (d, 3H,
J ¼ 6.9 Hz, CH3), 1.30 (s, 3H, CH3), 1.42 (s, 3H, CH3), 3.71 (s, 3H,
OCH3), 3.76e3.83 (m, 1H, H-50), 3.84e3.92 (m, 2H, H-1, H-50),
3.93e4.04 (m, 1H, H-2), 4.06e4.13 (m, 1H, H-40), 4.38 (d, 1H,
J ¼ 16.1 Hz, NCH2Ph), 4.48e4.59 (m, 2H, 1HeOCH2Ph, 1HeNCH2Ph),
4.76 (d, 1H, d, J ¼ 11.3 Hz, OCH2Ph), 7.14e7.36 (m, 10H, Ph); 13C NMR
4.1.10. 4.1.10. Methyl {(1S,2R)-1-(benzyloxy)-1-[(40R)-20,2'-
dimethyl-10,3'-dioxolan-4'-yl]-3-iodopropan-2-yl}carbamate (17)
To a solution of 7 (0.24 g, 0.71 mmol) in a mixture of 3:1 Et2O/
MeCN (7.2 mL) were successively added Ph3P (0.28 g, 1.08 mmol)
and imidazole (71.3 mg, 1.07 mmol), and the resulting solution was
stirred at room temperature for 15 min before addition of I2 (0.22 g,
1.78 mmol). After stirring for another 1 h, the mixture was washed
with a saturated Na2S2O3 solution (35 mL) and then extracted with
CH2Cl2 (2 ꢂ 50 mL). The combined organic layers were dried over
Na2SO4, the solvent was removed under reduced pressure, and the
residue was purified by flash chromatography on silica gel (n-
hexane/ethyl acetate, 5:1) to afford 0.27 g (85%) of compound 17 as
white crystals; mp 65e66 ꢁC (recrystallized from n-hexane/ethyl
(100 MHz, CDCl3, 60 ꢁC):
d 14.6 (CH3), 25.1 (CH3), 26.5 (CH3), 49.0
(NCH2Ph), 52.6 (OCH3), 54.3 (CH, C-2), 65.5 (CH2, C-50), 74.9
(OCH2Ph), 76.9 (CH, C-40), 80.8 (CH, C-1), 108.8 (Cq), 127.0 (CHPh),
127.7 (CHPh), 127.8 (2 ꢂ CHPh), 128.3 (3 ꢂ CHPh), 128.4 (3 ꢂ CHPh),
138.5 (Ci), 139.2 (Ci), 157.0 (C]O). Anal. Calcd for C24H31NO5: C,
69.71; H, 7.56; N, 3.39. Found: C, 69.78; H, 7.50; N, 3.44.
4.1.13. 4.1.13. Methyl benzyl[(2S,3S,4R)-3-(benzyloxy)-4,5-
dihydroxypentan-2-yl]carbamate (6)
acetate); [
a]
27 ꢀ 7.22 (c 0.62, CHCl3). IR ymax 3365, 2984, 2912, 1702,
p-TsOH (16.6 mg, 0.08 mmol) was added to a solution of 19
(0.18 g, 0.44 mmol) in MeOH (6.8 mL) and the mixture was stirred
at room temperature for 6 h. After neutralization with Et3N, the
solvent was removed in vacuo, and the residue was subjected to
flash chromatography on silica gel (n-hexane/ethyl acetate, 1:2) to
D
1515, 1454, 1370, 1328, 1226, 1157, 1093, 1070, 1022 cmꢀ1; 1H NMR
(400 MHz, CDCl3): d 1.35 (s, 3H, CH3), 1.44 (s, 3H, CH3), 3.43 (dd, 1H,
J ¼ 10.0 Hz, J ¼ 3.3 Hz, H-3), 3.51 (dd, 1H, J ¼ 10.0 Hz, J ¼ 3.9 Hz, H-
3), 3.66e3.75 (m, 5H, OCH3, H-1, H-2), 3.93 (app. t, 1H, J ¼ 7.3 Hz, H-
50), 4.08 (app. t,1H, J ¼ 7.3 Hz, H-50), 4.18e4.25 (m,1H, H-40), 4.69 (d,
1H, J ¼ 11.0 Hz, OCH2Ph), 4.81 (d, 1H, J ¼ 11.0 Hz, OCH2Ph), 4.95 (d,
1H, J ¼ 5.5 Hz, NH), 7.25e7.40 (m, 5H, Ph); 13C NMR (100 MHz,
afford 0.14 g (86%) of compound 6 as a colourless oil; [
a
]
26 ꢀ 25.4 (c
D
0.37, CHCl3). IR ymax 3419, 3029, 2952, 1671, 1451, 1403, 1240, 1213,
1046, 1027 cmꢀ1 1H NMR (400 MHz, CDCl3, 60 ꢁC):
; d 1.23 (d, 3H,
CDCl3):
d
10.2 (CH2, C-3), 25.1 (CH3), 26.5 (CH3), 52.5 (OCH3 or CH,
J ¼ 7.0 Hz, CH3), 3.63e3.75 (m, 6H, OCH3, 2 ꢂ H-5, H-4), 3.76e3.81
(m, 1H, H-3), 4.05e4.15 (m, 1H, H-2), 4.45e4.48 (m, 2H, NCH2Ph),
4.54 (d, 1H, J ¼ 11.3 Hz, OCH2Ph), 4.67 (d, 1H, J ¼ 11.3 Hz, OCH2Ph),
C-2), 52.6 (OCH3 or CH, C-2), 66.0 (CH2, C-5ˈ), 74.5 (OCH2Ph), 76.0
(CH, C-40), 79.5 (CH, C-1), 109.4 (Cq), 128.0 (2 ꢂ CHPh), 128.5
(3 ꢂ CHPh), 137.7 (Ci), 156.2 (C]O). Anal. Calcd for C17H24INO5: C,
45.45; H, 5.38; N, 3.12. Found: C, 45.52; H, 5.34; N, 3.16.
7.14e7.35 (m, 10H, Ph); 13C NMR (100 MHz, CDCl3, 60 ꢁC):
d 14.6
(CH3), 49.3 (NCH2Ph), 52.7 (OCH3), 54.1 (CH, C-2), 63.4 (CH2, C-5),
72.3 (CH, C-4), 74.4 (OCH2Ph), 83.2 (CH, C-3), 127.1 (2 ꢂ CHPh), 127.9
(5 ꢂ CHPh), 128.5 (3 ꢂ CHPh), 138.1 (Ci), 139.0 (Ci), 157.4 (C]O). Anal.
Calcd for C21H27NO5: C, 67.54; H, 7.29; N, 3.75. Found: C, 67.48; H,
7.34; N, 3.69.
4.1.11. 4.1.11. Methyl {(1S,2S)-1-(benzyloxy)-1-[(10R)-20,2'-
dimethyl-10,3'-dioxolan-4'-yl]propan-2-yl}carbamate (18)
To a solution of 17 (0.25 g, 0.56 mmol) in MeOH (45 mL) was
added 10% Pd/C (0.42 g) and the resulting suspension was then
treated with Et3N (0.41 mL, 2.94 mmol). The mixture was stirred for
1 h at room temperature under an atmosphere of hydrogen. After
completion of the reaction, the catalyst was removed by filtration
through a small pad of Celite, and the filtrate was concentrated.
Chromatography of the residue on silica gel (n-hexane/ethyl ace-
4.1.14. Methyl benzyl[(2S,3R,Z)-3-(benzyloxy)octadec-4-en-2-yl)
carbamate (Z)-20 and methyl benzyl[(2S,3R,E)-3-(benzyloxy)
octadec-4-en-2-yl)carbamate (E)-20
A solution of 6 (0.104 g, 0.28 mmol) in MeOH (0.6 mL) was
treated with a solution of NaIO4 (72 mg, 0.34 mmol) in H2O (0.3 mL)
and stirring was continued for 30 min at room temperature. After
completion of the reaction, the insoluble parts were filtered off, and
the filtrate was concentrated in vacuo. The residue was washed
with CH2Cl2 (2 ꢂ 5 mL), the combined organic layers were dried
over Na2SO4, and the solvent was evaporated. The obtained crude
product was used immediately in the next reaction without further
purification.
tate, 4:1) gave 0.17 g (96%) of compound 18 as a colourless oil;
27
[
a]
ꢀ 28.4 (c 0.45, CHCl3). IR ymax 3331, 2984, 2935, 1701, 1525,
D
1454, 1370, 1222, 1068 cmꢀ1; 1H NMR (400 MHz, CDCl3):
d 1.16 (d,
3H, J ¼ 6.9 Hz, CH3), 1.34 (s, 3H, CH3), 1.45 (s, 3H, CH3), 3.59e3.69
(m, 4H, OCH3, H-1), 3.86e3.92 (m, 1H, H-50), 3.96e4.12 (m, 3H, H-2,
H-50, H-40), 4.61 (d, 1H, J ¼ 11.5 Hz, OCH2Ph), 4.65e4.76 (m, 2H,
OCH2Ph, NH), 7.25e7.39 (m, 5H, Ph); 13C NMR (100 MHz, CDCl3):

ꢀ
ꢁ
M. Fabisíkova et al. / Carbohydrate Research 435 (2016) 26e36
35
To
a
solution of 1,1,1,3,3,3-hexamethyldisilazane (190
m
L,
mp not reported; lit [11h]. mp 65e67 ꢁC; lit [11i]. mp 76e77 ꢁC; lit
26
0.9 mmol) in dry THF (0.8 mL) was added n-BuLi (0.56 mL,
0.9 mmol, ~1.6 M solution in n-hexane) at room temperature. The
solution of LHMD such generated was treated with the known salt 5
[17] (0.5 g, 0.9 mmol) and the resulting dark mixture was stirred for
5 min at the same temperature. Then, a solution of the crude
aldehyde (95 mg, 0.28 mmol) in dry THF (0.8 mL) was added and
stirring was continued for 30 min. After completion of the reaction
(judged by TLC), the mixture was washed with a saturated NH4Cl
solution (5 mL). The organic layer was separated, and the aqueous
phase was extracted with further portions of EtOAc (2 ꢂ 10 mL). The
combined organic extracts were dried over Na2SO4, the solvent was
removed in vacuo, and the residue was chromatographed on silica
gel (n-hexane/ethyl acetate, 25:1) to give 0.129 g (88%) of a mixture
of alkenes 20. Repeated chromatography on silica gel (n-hexane/
ethyl acetate, 25:1) afforded only (Z)-20 in pure form as an
analytical sample.
[11j]. mp 65e67 ꢁC; [
a
]
þ 16.5 (c 0.20, CHCl3); lit [1b,9,11a-
D
d,11f,11h-j]. (see Table 3). IR ymax 2956, 2913, 2847, 1573, 1465,
1374, 1043 cmꢀ1 1H NMR (400 MHz, CD3OD):
;
d
0.87 (t, 3H,
J ¼ 6.6 Hz, CH3-18), 1.03 (d, 3H, J ¼ 6.6 Hz, CH3-1), 1.18e1.39 (m,
28H, 14 ꢂ CH2), 2.76e2.82 (m, 1H, H-2), 3.38e3.42 (m, 1H, H-3); 13
C
NMR (100 MHz, CD3OD):
d 14.5 (CH3, C-18), 16.8 (CH3, C-1), 23.8
(CH2), 27.3 (CH2), 30.5 (CH2), 30.8 (CH2), 30.9 (CH2), 33.1 (CH2), 34.0
(CH2), 52.2 (CH, C-2), 76.1 (CH, C-3). Anal. Calcd for C18H39NO: C,
75.72; H, 13.77; N, 4.91. Found: C, 75.77; H, 13.81; N, 4.87.
4.1.17. X-ray techniques
Single crystals of 14 suitable for X-ray diffraction were obtained
from Et2O by slow evaporation at room temperature. The in-
tensities were collected at 173 K on an Oxford Diffraction XCalibur2
CCD diffractometer using Mo K
a
radiation (
l
¼ 0.71073 Å). Selected
crystallographic and other relevant data for compound 14 are listed
in Table 2. The structure was solved by direct methods [23]. All non-
hydrogen atoms were refined anisotropically by full-matrix least
squares calculations based on F [2,23] All hydrogen atoms were
included in calculated positions as riding atoms, with SHELXL97 [23]
defaults. The PLATON [24] programme was used for structure
analysis and molecular and crystal structure drawings.
Isomer (Z)-20: colourless oil; [
a]
26 ꢀ 19.6 (c 0.49, CHCl3). IR ymax
D
2922, 2852, 1698, 1496, 1452, 1213, 1062 cmꢀ1; 1H NMR (400 MHz,
CDCl3, 60 ꢁC):
d
0.88 (t, 3H, J ¼ 6.8 Hz, CH3), 1.22 (d, 3H, J ¼ 6.9 Hz,
CH3), 1.25e1.37 (m, 22H, 11 ꢂ CH2), 1.99e2.14 (m, 2H, CH2), 3.66 (s,
3H, OCH3), 3.82e3.91 (m,1H, H-2), 4.24 (d,1H, J ¼ 11.7 Hz, OCH2Ph),
4.37e4.56 (m, 4H, OCH2Ph, 2HeNCH2Ph, H-3), 5.21e5.26 (m, 1H, H-
4), 5.65 (app. td, 1H, 11.0 Hz, J ¼ 7.4 Hz, H-5), 7.16e7.32 (m, 10H, Ph);
13C NMR (100 MHz, CDCl3, 60 ꢁC):
d
14.0 (2 ꢂ CH3, C-1, C-18), 22.7
4.1.18. Supplementary data
(CH2), 27.9 (CH2), 29.3 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2), 29.8
(CH2), 31.9 (CH2), 50.1 (NCH2Ph), 52.3 (OCH3), 57.1 (CH, C-2), 70.4
(OCH2Ph), 76.8 (CH, C-3), 126.8 (CHPh), 127.4 (CHPh), 127.6 (CH, C-4),
128.2 (4 ꢂ CHPh), 128.3 (4 ꢂ CHPh), 135.5 (CH, C-5), 138.9 (Ci), 139.5
(Ci), 156.6 (C]O). Anal. Calcd for C34H51NO3: C, 78.26; H, 9.85; N,
2.68. Found: C, 78.32; H, 9.80; N, 2.64.
Complete crystallographic data for the structural analysis have
been deposit with the Cambridge Crystallographic Data Centre,
CCDC No. 1456896 for compound 14. These data can be obtained
free of charge from the Director, Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: þ44 1223
336033; e-mail: deposit@ccdc.cam.ac.uk or via: www.ccdc.cam.ac.
uk).
4.1.15. 4.1.15. Methyl [(2S,3R)-3-hydroxyoctadecan-2-yl]carbamate
(21)
4.1.18.1. DFT calculations. A thorough conformational search was
performed on all transition states, although only the lowest energy
conformers are discussed herein. The potential energy surface
scans were used to explore the conformational space of transition
states TS1 and TS2 with the constrained transition bond lengths
CeN and CeS. The systematic conformational search was per-
formed using semiempirical PM3 methodology. The conformers
within a 3 kcal/mol energy range were then optimized using B3LYP/
6-31G(d) for a more accurate description of the conformer distri-
bution. The low energy conformers of the transition states were
later fully optimized using B3LYP/6-31G(d,p) with the Jaguar 7.7
program [27]. The potential energy surface scans were used to
explore the conformational space of the reactant and products
(thiocyanate 8 and isothiocyanates 14 and 15) with the later full
optimization using the same level of theory; however we only
focused on the energy differences between TSs as being crucial for
the explanation of the observed diastereoselectivity. The nature of
the vacuum B3LYP transition states was verified with frequency
calculations, yielding only one large imaginary frequency. Har-
monic zero-point energy corrections at B3LYP/6-31G(d,p) obtained
from the frequency calculations of the vacuum transition states
were applied to the transition-state energies. Single-point energies
were computed by the M06-2X density functional method and the
cc-pvTZ basis set. The solvent effect was taken into account via a
single-point calculation in a dielectric continuum representing n-
heptane as the solvent. A standard PBF solvation model was applied
as implemented in Jaguar 7.7 program [27].
To a solution of 20 (0.11 g, 0.22 mmol) in dry MeOH (6.2 mL) was
added successively 10% Pd/C (59 mg) and 20% Pd(OH)2/C (59 mg)
and the resulting suspension was stirred at 60 ꢁC for 48 h under an
atmosphere of hydrogen. After cooling to room temperature, the
catalyst was filtered through a small pad of Celite, and the solvent
was evaporated. Chromatography of the residue on silica gel (n-
hexane/ethyl acetate, 5:1) furnished 52 mg (72%) of compound 21
as white crystals; mp 101e103 ꢁC (recrystallized from n-hexane/
ethyl acetate); [
a
]
26 ꢀ 4.6 (c 0.26, CHCl3). IR ymax 3322, 2914, 2847,
D
1696, 1541, 1464, 1285, 1243, 1094, 1047 cmꢀ1; 1H NMR (400 MHz,
CDCl3):
d
0.88 (t, 3H, J ¼ 6.8 Hz, CH3-18), 1.09 (d, 3H, J ¼ 6.8 Hz, CH3-
1), 1.17e1.35 (m, 26H, 13 ꢂ CH2), 1.33e1.44 (m, 2H, CH2), 3.62e3.79
(m, 5H, H-2, H-3, OCH3), 4.99 (d, 1H, J ¼ 5.6 Hz, NH); 13C NMR
(100 MHz, CDCl3):
d 14.1 (CH3, C-1, CH3, C-18), 22.7 (CH2), 26.0
(CH2), 29.3 (CH2), 29.6 (CH2), 29.7 (CH2), 31.9 (CH2), 33.5 (CH2), 50.9
(CH, C-2), 52.1 (OCH3), 74.2 (CH, C-3), 156.8 (C]O). Anal. Calcd for
C
20H41NO3: C, 69.92; H, 12.03; N, 4.08. Found: C, 69.97; H, 11.98; N,
4.12.
4.1.16. 4.1.16. (2S,3R)-2-Aminooctadecan-3-ol (1)
A solution of 21 (52 mg, 0.15 mmol) in EtOH (3 mL) was treated
with 2 M NaOH (3 mL) and the resulting mixture was stirred and
heated under reflux for 1 h. After completion of the reaction, the
mixture was concentrated under reduced pressure, and the ob-
tained residue was washed with CH2Cl2 (2 ꢂ 5 mL). The combined
organic layers were dried over Na2SO4, the solvent was evaporated,
and the crude product was purified by flash chromatography
through a short column of silica gel (MeOH/CH2Cl2, 1:1). This pro-
cedure yielded 35 mg (82%) of the crystalline compound 1; mp
68e70 ꢁC; lit [9]. mp 65e66 ꢁC; lit [11a]. mp 64.5e66 ꢁC; lit [11b].
mp 115e116 ꢁC; lit [11c]. 65e67 ꢁC; lit [11d]. mp 67e68 ꢁC; lit [11f].
4.2. Antiproliferative/cytotoxic activity
4.2.1. Cell culture
The following human cancer cell lines were used for this study:

ꢀ
ꢁ
36
M. Fabisíkova et al. / Carbohydrate Research 435 (2016) 26e36
4579e4588.
MCF-7(breast cancer cells MDA-MB-231 (breast cancer cells), Jurkat
(human acute T-lymphoblastic leukaemia), HCT-116 (human colon
carcinoma), Caco-2 (human colon carcinoma) and HeLa (cervical
adenocarcinoma), and non-cancerous cell line NiH 3T3 (mouse fi-
broblasts). MCF-7, HCT-116, Caco-2, HeLa and Jurkat cells were
maintained in RPMI 1640 medium. MDA-MB-231 and NiH 3T3 cell
lines were maintained in growth medium consisting of high
glucose Dulbecco's Modified Eagle's Medium. Both of these media
were supplemented with Glutamax, and with 10% (V/V) foetal calf
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mLꢀ1), and streptomycin
ꢁ
serum, penicillin (100 IU
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(100 mg ꢂ mLꢀ1) (all from Invitrogen, Carlsbad, CA USA), in the
atmosphere of 5% CO2 in humidified air at 37 ꢁC. Cell viability,
estimated by the tryptan blue exclusion, was greater than 95%
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the colorimetric microculture assay with the MTT endpoint [25,26].
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 cultꢀu4re
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medium containing tested chemicals at final concentrations 10
‒
7438e7448;
10ꢀ6 mol ꢂ Lꢀ1. After 72 h incubation, 10
m
L of MTT (5 mg ꢂ mLꢀ1
)
(f) K. Xu, G. Lai, Z. Zha, S. Pan, H. Chen, Z. Wang, Chem.eEur. J. 18 (2012)
12357e12362;
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7223e7233;
were added into each well. After an additional 4 h, during which
insoluble formazan was formed, 100
mL of 10% (m/m) sodium
dodecylsulfate was 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
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(b) E. Abraham, S.G. Davies, N.L. Millican, R.L. Nicholson, P.M. Roberts,
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Acknowledgements
The present work was supported by the Grant Agency (No. 1/
0168/15 and No. 1/0398/14) of the Ministry of Education, Slovak
Republic. It was also supported by the Slovak Research and
Development Agency (No. APVV-14-0883), VVGS-2014-172 and by
(d) H.R. Vulupala, A. Sajja, R. Bantu, L. Nagarapu, Curr. Org. Chem. 19 (2015)
2040e2045;
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J.E. Thomson, Tetrahedron Lett. 57 (2016) 1270e1272.
[16] K. Stankova, M. Martinkova, J. Gonda, M. Bago, M. Pilatova, G. Gonciova,
Tetrahedron Asymmetry 26 (2015) 1394e1407.
ꢀ
the project MediPark Kosice: 26220220185 supported by Opera-
ꢁ
ꢁ
ꢁ
ꢁ
€
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tional Programme Research and Development (OP VaV-2012/2.2/
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08-RO, contract No. OPVaV/12/2013). We thank to Dr. Juraj Kuchar
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(P. J. Safarik University, Kosice) for his assistance in X-ray
measurements.
(b) G.S. Rao, N. Sudhakat, B.V. Rao, S.J. Basha, Tetrahedron Asymmetry 21
(2010) 1963e1970.
Appendix A. Supplementary data
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Supplementary data related to this article can be found at http://
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