Synthesis and in vitro biological evaluation of 3-amino-3-deoxydihydrosphingosines and their analogues

Article abstract of DOI:10.1016/j.tet.2019.130803

The stereoselective synthesis of the 3-amino-3-deoxydihydrosphingosines and their isomeric analogues from dimethyl L-tartrate is described by means of [3,3]-sigmatropic rearrangements and the cross metathesis reaction as a cornerstone of the developed str

Full text of DOI:10.1016/j.tet.2019.130803

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Synthesis and in vitro biological evaluation of 3-amino-3-deoxydihydrosphingosines
and their analogues
Jana Špaková Raschmanová, Miroslava Martinková, Jozef Gonda, Martina Bago
Pilátová, Juraj Kuchár, Dávid Jáger
PII:
S0040-4020(19)31207-4
https://doi.org/10.1016/j.tet.2019.130803
TET 130803
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 29 September 2019
Revised Date: 12 November 2019
Accepted Date: 13 November 2019
Please cite this article as: Raschmanová JanaŠá, Martinková M, Gonda J, Pilátová MB, Kuchár J,
Jáger Dá, Synthesis and in vitro biological evaluation of 3-amino-3-deoxydihydrosphingosines and their
analogues, Tetrahedron (2019), doi: https://doi.org/10.1016/j.tet.2019.130803.
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Graphical Abstract
Synthesis and in vitro evaluation of 3-amino-3-deoxydihydrosphingosines and their analogues
Jana Špaková Raschmanová , Miroslava Martinková*, Jozef Gonda, Martina Bago Pilátová, Juraj Kuchár and Dávid Jáger

Synthesis and in vitro biological evaluation of 3-amino-3-
deoxydihydrosphingosines and their analogues
a
a,
Jana Špaková Raschmanová , Miroslava Martinková , Jozef Gonda a,†, Martina Bago
Pilátová b, Juraj Kuchár c, Dávid Jáger d
aInstitute of Chemical Sciences, Department of Organic Chemistry, P.J. Šafárik University, Moyzesova
11, 040 01 Košice, Slovak Republic
bInstitute of Pharmacology, Faculty of Medicine, P.J. Šafárik University, SNP 1, 040 66 Košice,
Slovak Republic
cInstitute of Chemical Sciences, Department of Inorganic Chemistry, P.J. Šafárik University,
Moyzesova 11, 040 01 Košice, Slovak Republic
dInstitute of Geotechnics, Slovak Academy of Sciences, Watsonova 45, 040 01 Košice, Slovak Republic
†This work is dedicated to the memory of Professor Jozef Gonda (1957-2019)
ARTICLE INFO
Article history:
Received
Accepted
Available online
Keywords:
Sphingoid base; [3,3]-Sigmatropic rearrangements; Olefin cross-metathesis; Antiproliferative
activity
ABSTRACT
The stereoselective synthesis of the 3-amino-3-deoxydihydrosphingosines and their isomeric
analogues from dimethyl L-tartrate is described by means of [3,3]-sigmatropic rearrangements
and the cross metathesis reaction as a cornerstone of the developed strategy. The
configuration of two newly created stereocentres was unambiguously assigned via a single
crystal X-ray analysis of the complex structure of di-tert-butyl [(2R,3R,4S)-1,2-
Corresponding author.
e-mail address: miroslava.martinkova@upjs.sk (M. Martinková).
1

dihydroxynonadecane-3,4-diyl]dicarbamate. The prepared unusual sphingoid bases were
evaluated regarding their capacity to alter the viability of cancer cell lines.
1. Introduction
Sphingolipids (SLs) represent unique components of all eukaryotic cells. Besides their pivotal
role in membrane construction, their metabolic pathways deliver compounds that participate
in various significant cellular signalling processes, such as angiogenesis, cell proliferation,
apoptosis, differentiation, inflammation and adhesion.1 On the other hand, recent studies have
revealed that defects in sphingolipid metabolism lead to several human diseases.1b-f,2 This
wide family of lipids shares common structural backbones, including
D-erythro-sphingosine
1,3a,3d-e -erythro-dihydrosphingosine (sphinganine) 23c and -ribo-phytosphingosine 33b,3d-e
D
D
(Fig. 1). It should be noted that the mentioned sphingoid bases exhibit an interesting
biological profile. Both sphingosine 1 and sphinganine 2 were found to inhibit protein kinase
C (PKC).4 Dihydrosphingosine 2 and its unnatural
D/L-threo-diastereoisomers, the general
structure of which is illustrated by safingol 43c,5 (Fig. 1), are well-known sphingosine kinase
inhibitors (SphK).1f Sphingosine kinases (SphKs)1f,6 are enzymes that catalyze the conversion
of 1 to sphingosine-1-phosphate (S1P). In addition to dihydrosphingosine 2 and its further
threo-congeners, 3 also belongs to the family of SphK inhibitors.6
D-erythro-Sphingosine 1
acts as a metabolic switch between ceramide (Cer) and S1P. These two structures are crucial
regulators of cell functions. The former initiates an apoptotic process, while the latter is a
recognized promoter of cell survival.1c,7
Figure 1. Representative natural sphingoid bases 1-3 and their synthetic analogues.
2

On the other hand,
D-ribo-phytosphingosine 3 was identified as a heat-stress signalling
molecule in yeast.8 N-Acyl-linked derivatives of 3 (also called phytoceramides) have been
found in large quantities in the extracellular spaces of the epidermis (stratum corneum) in
humans.9 Furthermore, phytosphingosine 3 exhibits a pro-apoptotic effect against different
cancer cell lines.10 Kim’s11 group revealed the cytotoxic activity of 3 on three different human
malignant cells (PC-3, HCT-116 and A-549). Due to the potent biological properties of
naturally occurring sphingoid bases, there has been extensive focus on the construction of
various analogues of 1-3 in recent years with the aim of finding appropriate candidates or lead
compounds for the design of novel sphingolipid metabolism modulators. Among the
synthesized derivatives (for some examples, see Fig. 1), for example, the following stand out,
isophytosphingosines 512,13 and isomeric sphinganines 6,13b,14,15 which display remarkable
cytotoxicity; fluorinated triazole-containing analogues16 of 1 (up to 16 compounds depicted
by the general structure 7) with SphK’s inhibitory activity; amide-linked derivatives17 of 1
and 2 substituted with different types of nucleophiles at the C-1 position, with the ability to
block glucosylceramide synthase and yeast inositol phosphorylceramide synthase; and last but
not least, 3-amino-3-deoxy analogues18 of 2 and 3.
Cancer is the second leading cause of death worldwide. Modulation of sphingolipid
biosynthesis and metabolism is expected to be one of the promising approaches for therapy of
the aforementioned disease. Our group has been intensively dealing with the stereoselective
construction of various types of sphingoid base-like compounds13,15,19 having an anticancer
profile, starting from simple and available chirons. As part of our continuing studies on the
synthesis of biologically active sphingolipid related compounds, we herein report the
preparation of the cytotoxic 3-amino-3-deoxydihydrosphingosines 10 and 11 along with their
analogues 12-15 from dimethyl L-tartrate as the chiral pool material. It should be noted that
we recently elaborated a small library of cytotoxic C17-sphinganine analogues 613b,15 (Fig. 1).
In light of our recent synthetic development,13b,15 we set out to extend the panel of targeted
dihydrosphingosine and sphingosine analogues, including diamino congeners. We also bring
improved diastereoselectivity of the aza-Claisen rearrangement of thiocyanates (E/Z)-17 (vide
infra) with an incorporated bulky cyclohexylidene ketal protecting group in comparison with
the previous work, which used the corresponding acetonide.15 Moreover, to the best of our
knowledge, the present study will be the first report in the literature on the
antiproliferative/cytotoxic activities of sphingoid bases with the vicinal diamino motif. Such
production of sphingolipid analogue libraries can be useful for structure-activity studies in
biological targets.
3

2. Results and discussion
2.1. Chemistry
The general synthetic strategy towards modified sphingoid bases 10-15 is outlined in Scheme
1. For all target compounds, the introduction of the aliphatic chain was planned to be realized
at a later stage in the synthesis via an olefin cross-metathesis (OCM) reaction. It was
anticipated that the [3,3]-sigmatropic rearrangements on the allylic substrates E/Z-16, E/Z-17
and 18 would create the expected synthons 19-20 and 21-22, respectively. Imidates E/Z-16
and thiocyanates E/Z-17 can be readily obtained from dimethyl L-tartrate. On the other hand,
rhodanide 18 would be synthesized from the major template 19 through a short sequence
involving an ozonolysis, Wittig olefination and reduction.
O
NHBoc
SCN
O
O
O
[3,3]-
[3,3]-
O
O
O
O
R
NHBoc
21 22
NHBoc
NHBoc
19 20
16
E/Z- , R = O(CNH)CCl3
18
/
/
17
E/Z- , R = SCN
OCM
OCM
dimethyl
L-tartrate
NH2.HCl
C14H29
OH C14H29
and
OH
C13H27
HO
NH2.HCl
10 11
OH NH2.HCl
12 13
OH NH2.HCl
14 15
,
,
,
Scheme 1. Retrosynthetic analysis towards modified sphingoid bases 10-15.
As seen in Scheme 2, the starting dimethyl
L-tartrate was transformed to the known protected
L
-threitol 23 (74%, over two steps) according to the literature protocol.20 Then the remaining
hydroxyl groups of 23 were protected with cyclohexanone using catalytic amounts of p-TsOH
to give 2421 in a yield of 85%. Compound 24 was converted into diol 25 (92%) by catalytic
hydrogenolysis. The oxidative fragmentation of 25, followed by Horner-Wadsworth-Emmons
olefination with (EtO)2P(O)CH2CO2Et and NaH, delivered α,β-unsaturated ester (E)-2622
exclusively in 89% yield. On the other hand, the Wittig reaction with a stable ylide reagent
afforded a mixture of isomers 26 (E:Z = 39:61, combined yield of 92%) with a mild
22
prevalence of (Z)-26 (56%, >97:3 dr), [α]D ‒77.0 (c 0.28, CHCl3); for (E)-26 (36%, >97:3
4

21
dr), [α]D –31.9 (c 0.34, CHCl3). Column chromatography on silica gel furnished each
diastereoisomer in pure form.
It is known that Cinquini, Cozzi and co-workers22 prepared the corresponding methyl (R,Z)-3-
(1,4-dioxaspiro[4.5]decan-2-yl)acrylate, via the Still-Gennari protocol23 as the sole product. It
should be noted that this aforementioned procedure uses an exceedingly expensive reagent
(methyl 2-[bis(2,2,2-trifluoroethoxy)phosphoryl]acetate). Therefore, we decided to apply the
more available Wittig’s conditions (Ph3P=CHCO2Et, benzene) towards (Z)-26 (ethyl (R,Z)-3-
(1,4-dioxaspiro[4.5]decan-2-yl)acrylate), although the selectivity of the olefination was
moderate (Scheme 2).
The production of larger amounts of (Z)-26 then allowed the preparation of four allylic
substrates, (E)-16, (Z)-16, (E)-17 and (Z)-17 (vide infra), for the comparative study of
subsequent [3,3]-sigmatropic rearrangements.
OH
O
O
O
b
O
ref. 20
a
c and d
HO
dimethyl L-tartrate
OH
OH
CO2Et
OBn
23,20 74%
(over two steps)
OR
conditions c: (E)-
26 22
89%
,
24,21 R = Bn, 85%
conditions d: (E)-26:(Z)-26 =
25, R = H, 92%
39:61, 92%
Scheme 2. Reagents and conditions: (a) cyclohexanone, p-TsOH, toluene, rt; (b) H2, 10% Pd/C/20% Pd(OH)2/C
(1:1), EtOH, rt; (c) (i) NaIO4, CH2Cl2/H2O (2.5:1), rt; (ii) (EtO)2P(O)CH2CO2Et, NaH, THF, 0 °C; (d) (i) NaIO4,
CH2Cl2/H2O (2.5:1), rt; (ii) Ph3P=CHCO2Et, benzene, reflux, (E)-26 (36%), (Z)-26 (56%).
Reduction of (E)-26 and (Z)-26 with DIBAl-H provided the corresponding alcohols (E)-27
and (Z)-27 in 93% and 92% yields, respectively (Scheme 3). To continue the synthesis, our
attention focused on the Overman rearrangement of trichloroacetimidates (E)-16 and (Z)-16,
which were obtained from (E)-27 and (Z)-27 by the action of CCl3CN and DBU. The
microwave-promoted thermal rearrangement of both imidates (E)-16, (Z)-16 took place in o-
xylene in the presence of K2CO3.24 In an effort to shorten the reaction time and improve the
combined yield of rearranged products 28 and 29, the microwave-promoted thermal Overman
rearrangement of (E)-16 and (Z)-16 was tested at three temperatures: 150 °C, 170 °C and 190
°C. As seen in Table 1, the high temperature (190 °C, entries 3 and 6) was shown to be the
most optimal for very good production of the corresponding trichloroacetamides 28 and 29,
5

which, however, were isolated as an inseparable mixture of diastereoisomers due to their
identical Rf values.
The observed diastereoselectivity in the thermal rearrangement of (E)-16 showed that both
faces of its allylic system are about equally preferred. In the case of (Z)-16, the geometry of
the double bond in an allylic trichloroacetimidate system should be an important factor for the
facial selectivity to prefer the production of the 1,2-anti-disatereoisomer 28 (Table 1, entries
4-6), and the energy differences between the transition states are key for explaining the
observed diastereoselectivity.
Scheme 3. Reagents and conditions: (a) DIBAl-H, CH2Cl2, ‒50 °C; (b) CCl3CN, DBU, CH2Cl2, 0 °C; (c) Table
1.
Table 1. Overman rearrangement of imidates (E)-16 and (Z)-16
Entry
Imidate
Conditionsa,b
Time (h)
Ratioc
28:29
49:51
50:50
50:50
80:20
75:25
74:26
84:16
‒
Yieldd (%)
1
2
3
4
5
6
7
8
(E)-16
(E)-16
(E)-16
(Z)-16
(Z)-16
(Z)-16
(E)-16
(Z)-16
MW, 150 °Ca
MW, 170 °Ca
MW, 190 °Ca
MW, 150 °Ca
MW, 170 °Ca
MW, 190 °Ca
∆, 40 °Cb
8
60
67
90
60
73
85
40
3.5
0.33
19
4
1
1
∆, 70 °Cb
2
‒
e
a In o-xylene, in the presence of K2CO3.
b In toluene, in the presence of PdCl2(MeCN)2 and p-benzoquinone.
c Ratio in the crude reaction mixtures determined by 1H NMR.
d Isolated combined yields.
e Decomposition.
Palladium(II)-catalyzed Overman rearrangement of (E)-16 was carried out according to
Sutherland’s protocol25 (PdCl2(MeCN)2, p-benzoquinone26) to afford a 84:16 diastereomeric
ratio of the anti- and syn-amides 28 and 29, respectively (entry 7, Table 1). The lower
combined yield (40%) was due to the Lewis acidity of Pd(II), which induced the hydrolysis of
the cyclohexylideneketal fragment27 of (E)-16. Another competing pathway can be the
6

decomposition of the trichloroacetiminoyl group. The stereochemical outcome of the
catalyzed rearrangement of (E)-16 was in good agreement with the observations of Sutherland
and Swift27 and matched the erythro configuration of the vicinal aminoalcohol motif in the
major product 28. Upon applying Sutherland’s conditions to (Z)-16, only unidentified
decomposition products were generated in the reaction mixture (Table 1, entry 8).
Next, we explored the aza-Claisen rearrangement of thiocyanates (E)-17 and (Z)-17.
Incorporation of the –SCN group was accomplished by means of a nucleophilic substitution
after mesylation of the primary alcohol functionality of (E)-27 and (Z)-27 to provide the
corresponding products (E)-17 and (Z)-17 in 82% and 90% yields, respectively (Scheme 4).
With the aim of finding the optimal reaction conditions, both the thermally driven and
microwave-assisted [3,3]-sigmatropic rearrangements of (E)-17 and (Z)-17 were tested at
three different temperatures (50 °C, 70 °C and 90 °C) in n-heptane to produce the required
isothiocyanates 30 and 31 as readily separable diastereomeric mixtures. During these
experiments the starting material, either in the pure form as (E)-17 or (Z)-17, or as the
corresponding mixture (Z/E)-17, was also recovered in amounts of 10-71% (Table 2). As seen
in Table 2, high erythro-30/threo-31 selectivity along with a satisfying yield were obtained in
the case of (Z)-17 at 50 °C and 70 °C (Table 2, entries 8, 9 and 11, respectively). The major
isothiocyanate 30 is a kinetic reaction product, and the realization of the rearrangement at
lower temperature (50 °C) enabled control of the selectivity (Table 2 entries 8 and 9) while
still providing a satisfying yield (56% and 54%, respectively) of this transformation. The ratio
of the rearrangement was also influenced by the bulky cyclohexylidene moiety in comparison
to the isopropylidene group used in our previous study.15 Prolonged heating and higher
temperature were shown to be detrimental for the selectivity of the proceeding transformation
(Table 2, entries 6, 7 and 12) and revealed that the equilibrium of the studied reaction was
shifted to the thermodynamically more stable isothiocyanate 31 to decrease the 1,2-anti-
diastereoselectivity. On the other hand, it furnished a greater amount of the 1,2-syn-product
31 as the synthon for the preparation of the sphingoid bases 13 and 15. Analogously to our
recent work,15 the observed anti-vicinal stereochemistry in the [3,3]-sigmatropic
rearrangement of thiocyanates (E)-17 and (Z)-17 would also be explained by the results of
DFT calculations previously reported.15
7

Scheme 4. Reagents and conditions: (a) (i) MsCl, Et3N, CH2Cl2, 0 °C; (ii) KSCN, MeCN, rt; (b) Table 2.
Table 2. [3,3]-Sigmatropic rearrangement of thiocyanates (E)-17 and (Z)-17
Entry
Thiocyanate Conditionsa
Time (h)
Ratiob
30:31
81:19
75:25
81:19
74:26
72:28
72:28
72:28
94:6
95:5
90:10
91:9
75:25
85:15
Yieldc (%)
Isolated
thiocyanates (%)
1
2
3
4
5
6
7
8
(E)-17
(E)-17
(E)-17
(E)-17
(E)-17
(E)-17
(E)-17
(Z)-17
(Z)-17
(Z)-17
(Z)-17
(Z)-17
(Z)-17
ꢀ, 50 °C
∆, 50 °C
MW, 50 °C
∆, 70 °C
MW, 70 °C
ꢀ, 90 °C
MW, 90 °C
ꢀ, 50 °C
MW, 50 °C
ꢀ, 70 °C
MW, 70 °C
∆, 90 °C
MW, 90 °C
2.5
12
29
59
58
83
80
78
81
56
54
74
79
90
80
71d
37d
4.5
4.5
1.5
0.75
0.33
12
6
8
4
1.5
0.75
39d
17d
18d
17d
14d
42e
9
46e
10
11
12
13
22f E/Z = 87:13
21f E/Z = 99:1
10f E/Z = 75:25
16f E/Z = 67:33
a In n-heptane.
b Ratio in the crude reaction mixtures determined by 1H NMR.
c Isolated combined yields.
d Isolated (E)-17.
e Isolated (Z)-17.
f Isolated a mixture of thiocyanates (E/Z)-17. Determined by 1H NMR analysis.
Then the C-3 stereochemistry in the rearranged products 28-31 had to be assigned (Scheme
5). For this purpose, the conversion of the mixture of amides 28 and 29 (28:29 = 75:25,
determined by 1H NMR analysis) into known oxazolidin-2-ones15 33 (60%) and 34 (20%) was
achieved via removal of the ketal moiety, delivering an inseparable mixture of diols 32 (95%,
dr = 75:25) and a base induced ring-closure in 32. Their NMR characteristics, optical rotation
data and melting point were in accordance with those reported in the literature for the same
26
compounds; for 33: mp 102−104 °C; [α]D22 –8.1 (c 0.40, MeOH); lit.15 mp 102−105 °C; [α]D
–7.0 (c 0.37, MeOH); for 34: [α]D22 –85.1 (c 0.15, MeOH); lit.15 [α]D26 –87.5 (c 0.16, MeOH).
On the other hand, treatment of the threo-isothiocyanate 31 with p-TsOH in MeOH mediated
the spontaneous intramolecular addition of the liberated secondary alcohol to the –NCS group
to produce the known cyclic thiocarbamate 3515 in a yield of 96% (Scheme 5). The isolated
material had spectroscopic data, optical rotation and melting point in good agreement with
21
26
those reported; mp 130−132 °C, [α]D −158.3 (c 0.60, CHCl3); lit.15 mp 130−133 °C, [α]D
8

−144.6 (c 0.48, MeOH). These experiments revealed that both major products of the realized
rearrangements (compounds 28 and 30) are erythro-configured.
Scheme 5. Reagents and conditions: (a) p-TsOH, MeOH, 65 °C; (b) DBU, CH2Cl2, 0 °C→rt; (c) p-TsOH,
MeOH, rt.
Following the retrosynthetic analysis depicted in Scheme 1, the stage was now set for the
synthesis of the allylic substrate 18, starting from the major isothiocyanate 30 as an
appropriate synthon (Overman products 28 and 29 were not separable). Reaction of 30 with
bis(n-tributyltin) oxide (TBTO) and subsequent tert-butoxycarbonylation of the amino group
generated afforded N-Boc derivative 19 (78%, over two steps, Scheme 6). Ozonolysis of 19,
followed by Ph3P treatment, furnished an aldehyde, which was then subjected to HWE
olefination to afford (E)-α,β-unsaturated ester 36 (78%, over two steps) as the sole product.
Reduction of the ester functionality delivered allylic alcohol 37 (91%), which was converted
into the rhodanide 18 (87%, over two steps) using the same procedure as described for the
transformation of (E)-27 to (E)-17. The standard thermal reaction as well as the microwave
irradiation of 18 resulted in the formation of 3,4-syn- and 3,4-anti-diastereoisomer 38 and 39,
respectively (for the reaction conditions, combined yields and ratios, see Table 3). Column
chromatography then allowed the separation of both isothiocyanates 38 and 39.
9

Scheme 6. Reagents and conditions: (a) (i) TBTO, toluene, 60 °C; (ii) Boc2O, Et3N, CH2Cl2, rt; (b) (i) O3, EtOH,
‒78 °C, then Ph3P, CH2Cl2, rt; (ii) (EtO)2P(O)CH2CO2Et, NaH, THF, 0 °C; (c) DIBAl-H, BF3.OEt2, CH2Cl2, ‒78
°C; (d) (i) MsCl, Et3N, CH2Cl2, 0 °C; (ii) KSCN, MeCN, rt; (e) Table 3.
Table 3. [3,3]-Sigmatropic rearrangement of thiocyanate 18
Entry
Thiocyanate Conditionsa
Time (h)
Ratiob
38:39
84:16
85:15
81:19
83:17
78:22
79:21
77:23
79:21
Yieldc (%)
Isolated
18 (%)
43
53
11
25
11
26
15
1
2
3
4
5
6
7
8
ꢀ, 30 °C
14
51
41
79
71
81
72
82
81
18
18
18
18
18
18
18
18
MW, 30 °C
∆, 50 °C
6.5
2.5
1.25
1
0.5
1
MW, 50 °C
∆, 70 °C
MW, 70 °C
∆, 90 °C
MW, 90 °C
0.33
9
a In n-heptane.
b Ratio in the crude reaction mixtures determined by 1H NMR.
c Isolated combined yields.
The C-4 stereochemistry of 38 and 39 was assigned via a single crystal X-ray analysis (Fig. 2)
of the more advanced intermediate 44 (vide infra) and revealed that the major rearranged
product 38 has 3,4-syn-configuration.
10

Figure 2. ORTEP structure of 44 showing the crystallographic numbering.
Having confirmed a structure of the key templates 30, 31, 38 and 39, our focus was then
shifted to the synthesis of the final compounds 10-15 (Schemes 7 and 8). To construct 3-
amino-3-deoxydihydrosphingosines 10 and 11, the corresponding isothiocyanates 38 and 39
were, after treatment with TBTO, submitted to the reaction with Boc2O to furnish the
protected derivatives 21 (65%) and 22 (60%, in both cases over two steps). The olefin cross
metathesis reaction of 21 and 22 with pentadec-1-ene in the presence of second generation
Grubbs catalyst (CH2Cl2, reflux for 21 or toluene, 60 °C for 22) gave the (E)-configured
coupling product 40 (92%) and a mixture of olefins 41 (87%), with a high prevalence of (E)-
isomer. Subsequent reduction of the double bond under catalytic hydrogenation conditions
(H2, 5% Rh/Al2O3) led to the saturated products 42 (98%) and 43 (92%). Exposure of 42 and
43 to acid hydrolysis then resulted in cleavage of the cyclohexylidene ketal to provide diols
44 (74%) and 45 (78%). The modification of 44 and 45 into the diamino alcohols 46 (87%)
and 47 (83%) was achieved via an oxidation/reduction process. Finally, exposure of 46 and 47
to 6 M HCl furnished the expected sphingoid bases 10 (85%) and 11 (88%), respectively
11

(Scheme 7). As an additional determination of structure, we converted 10 into the known
acetylated derivative 48 (87%). The obtained material exhibited spectroscopic (1H and 13C
21
22
NMR) and optical rotation data ([α]D = ‒19.4, (c 0.3, CHCl3); lit.18 [α]D = ‒16.1 (c 1,
CHCl3)) consistent with those reported in the literature for the same compound.18 Melting
point values were found to show small differences (mp 100‒102 °C; lit.18 mp 115‒116 °C).
Scheme 7. Reagents and conditions: (a) (i) TBTO, toluene, 60 °C; (ii) Boc2O, Et3N, CH2Cl2, rt; (b) pentadec-1-
ene, Grubbs II, CH2Cl2, reflux or toluene, 60 °C; (c) H2, 5% Rh/Al2O3, EtOH or EtOH/THF (1:1), rt; (d) p-
TsOH, MeOH/H2O (20:1), 55 °C; (e) (i) NaIO4, MeOH/H2O, rt; (ii) NaBH4, EtOH, 0 °C; (f) 6 M aq HCl, 80 °C;
(g) Ac2O, pyridine, DMAP, rt.
A similar strategy as used in Scheme 7 was applied to the synthons 19 and 20 to obtain
isomeric analogues of 1 and 2 (compounds 12-15, Scheme 8). For this purpose, the minor
isothiocyanate 31 was modified into N-Boc protected derivative 20 (75%, over two steps)
according to the same reaction conditions employed for the conversion of 30 to 19 (see,
12

Scheme 6). The coupling of pentadec-1-ene with 19 and 20 was accomplished via cross-
metathesis to give (E)-olefin 49 (96%), and a separable mixture of the geometric isomers 50
(89%) with the dominant production of (E)-isomer. Column chromatography on silica gel
allowed the straightforward separation of both compounds to give pure (E)-50 (81%, >98:2
dr) and (Z)-50 (8%, >96:4 dr), respectively. Because of the overlap of proton signals in the 1H
NMR spectrum in the crude reaction mixture, we were unable to determine the E/Z-ratio. The
catalytic hydrogenation of 49 and (E)-50, followed by global deprotection of 51 (90%) and 52
(97.5%), delivered the isodihydosphingosines 12 and 13 in 90% and 83% yields, respectively
(Scheme 8). The corresponding isomeric sphingosines 14 (80%) and 15 (87%) were then
prepared from the coupling products 49 and (E)-50 after hydrolysis under acidic conditions.
Scheme 8. Reagents and conditions: (a) (i) TBTO, toluene, 60 °C; (ii) Boc2O, Et3N, CH2Cl2, rt; (b) pentadec-1-
ene, Grubbs II, CH2Cl2, reflux; (c) H2, 5% Rh/Al2O3, EtOH, rt; (d) 6 M aq HCl, 80 °C.
2.2. Antiproliferative/cytotoxic activity
13

A series of novel sphingoid bases 10-15 was evaluated for their in vitro cytotoxicity against
several different human cancer cell lines, such as MDA-MB-231 (mammary gland
adenocarcinoma), A-549 (non-small cell lung cancer), MCF-7 (mammary gland
adenocarcinoma), Caco-2 (human colon carcinoma) and HCT-116 (human colon carcinoma),
which were selected for compounds 12-15 (Table 4). A panel of four malignant cells ‒ PaTu
(human pancreatic adenocarcinoma), HeLa (human cervical adenocarcinoma), A2058 (human
melanoma cells), Jurkat (human acute T-lymphoblastic leukaemia) ‒ was used for all final
derivatives 10-15 (Table 5). Two types of non-malignant cell lines, specifically NiH 3T3
(mouse fibroblasts) and MCF-10A (human mammary epithelial cells) were also included. The
potency of the screened structures was determined by MTT assay with triplicate
experiments.28 Traditional anticancer substances cisplatin and etoposide (VP-16) were
included as a positive control, and the corresponding results are shown in Tables 4 and 5.
The cytotoxicity results (Table 5) revealed that compounds 10-15 displayed remarkable
antiproliferative activity against all the screened cell lines. Derivatives 12-15 were also
significantly active on MDA-MB-231, A-549, MCF-7, HCT-116 and Caco-2 cells, with the
IC50 values lower or comparable to that of cisplatin (Table 4).
Overall, all the tested derivatives were found to be more potent on leukaemia than on solid
tumour cell lines (compare Tables 4 and 5). Moreover, 3-amino-3-deoxydihydrosphingosine
10 (IC50 < 0.5 ꢁM for Jurkat, IC50 = 1.3 ꢁM for HeLa) demonstrates higher potency against
Jurkat and HeLa cells than commercially available etoposide (IC50 = 1.2 ꢁM for Jurkat, IC50 =
3.9 ꢁM for HeLa), respectively. Similarly, isomeric sphinganines 13 (IC50 = 1.3 ꢁM) and 14
(IC50 = 1.8 ꢁM) are at least 9-12 × more active against the Jurkat cell line than cisplatin (IC50
= 16.2 ꢁM). Unfortunately, as seen in Table 5, all synthesized sphingoid bases 10-15
demonstrate very low values of SI (selectivity index) across all cell lines. This suggests that
these compounds are equally or more toxic for non-malignant human mammary epithelial
cells MCF-10A. Similarly, compounds 12-15 showed powerful cytotoxicity for mouse
fibroblasts NiH 3T3 (Table 4). Therefore, the final sphingoid bases 10-15 could be further
transformed to reduce their toxicity and to enhance selectivity.
Table 4. Antiproliferative/cytotoxic activities of compounds 12-15 on five human cancer cell lines (MDA-MB-
231, A-549, MCF-7, HCT-116 and Caco-2) and non-malignant mouse fibroblasts NiH 3T3
Cell line, IC50a ± SD (µmol × L-1)
Compd
no.
MDA-
MB-231
6.9±0.4
A-549
MCF-7
8.2±1.6
HCT-116 Caco-2
3.3±0.6 6.3±2.1
NiH 3T3
<0.5
7.0±0.7
12
14

6.0±2.5
8.9±1.6
7.8±0.1
5.7±1.9
6.8±1.3
7.2±0.9
7.7±1.3
7.5±1.9
7.6±0.1
10.9±2.1
15.6±0.3
2.2±1.3
5.7±0.2
6.6±0.2
NT
5.5±2.3
8.3±2.0
7.7±0.6
NT
3.3±2.2
5.6±2.1
2.0±1.9
NT
13
14
15
VP-1629 21.2±4.2 14.3±4.2
cisplatin 17.5±0.5 9.5±0.2
15.3±0.5
15.2±0.3
20.87±0.3
a
The potency of compounds was determined using MTT assay after 72 h incubation of cells and given as IC50
(concentration of a tested compound that decreased the number of viable cells to 50% relative to untreated
control cells, see Section 4.2).
NT-not tested.
Table 5. Antiproliferative/cytotoxic activities of compounds 10-15 on four human cancer cell lines (PaTu, HeLa,
A2058, Jurkat) and non-malignant human mammary epithelial cells MCF-10A
Cell line, IC50a ± SD (µmol × L-1)
Compd
no.
PaTu
HeLa
A2058
Jurkat
<0.5
MCF-
10A
8.9±3.9
23.1±12.7 5.0±5.9
7.8±2.7
6.2±2.3
5.3±1.8
7.8±1.7
1.3±1.0
0.7±0.3
21.6±5.8 7.0±1.4
6.4±1.9
4.3±2.8
7.0±1.4
6.5±1.8
0.8±0.2
16.5±10.5
6.9±0.1
7.3±4.6
6.2±2.2
9.4±0.9
NT
10
11
12
13
14
15
6.1±1.4
5.9±2.2
6.7±1.2
4.1±2.5
13.1±0.2 NT
3.9±2.3 NT
2.4±1.0
1.3±0.9
1.8±2.0
4.2±2.2
16.2±0.6
1.2±1.5
cisplatin NT
VP-1629 NT
NT
a
The potency of compounds was determined using MTT assay after 72 h incubation of cells and given as IC50
(concentration of a tested compound that decreased the number of viable cells to 50% relative to untreated
control cells, see Section 4.2).
NT-not tested.
3. Conclusion
In summary, we successfully performed the diastereoselective construction of 3-amino-3-
deoxydihydrosphingosines 10-11 and their analogues 12-15 by means of [3,3]-sigmatropic
rearrangements of the allylic substrates (E/Z)-16, (E/Z)-17 and 18, and a late stage OCM
reaction. The corresponding stereochemistries at C-2 and C-3 in 10-11 and at C-3 in 12-15
were unambiguously assigned through crystallographic analysis of the complex structure 44.
The target compounds 10-15 were evaluated in vitro for their capacity to alter the viability of
the tested cancer cell lines. All prepared unusual sphingoid bases displayed cytotoxicity
against screened malignant cell lines with a potency higher or comparable to those of the
traditional anticancer agents cisplatin and etoposide. On the other hand, the observed strong
toxicity towards non-malignant cell lines requires demands for further studies aiming at the
construction of other analogues either with the alkyl chain modified by means of Grubbs
coupling or with the installed ceramide functionality. An additional study to improve the
activity profile of these compounds is already underway in our laboratory and progress will be
reported in due course.
15

4. Experimental section
4.1. Chemistry
4.1.1. General methods
Column chromatography was run on silica gel Kieselgel 60 (0.040‒0.063 mm, 230‒400 mesh,
Merck) under pressure, with solvents that had been distilled prior to use. Analytical thin layer
chromatography was performed on Merck silica gel 60 F254 plates and the visualization
utilized either UV light (254 nm) or spraying with a solution of phosphomolybdic acid, with
subsequent heating. NMR spectra were recorded on a Varian Mercury Plus 400 FT NMR
1
(400.13 MHz for H and 100.61 MHz for 13C) spectrometer or on a Varian Premium
COMPACT 600 (599.87 MHz for 1H and 150.84 MHz for 13C). For 1H, δ are given in parts
per million (ppm) either relative to TMS (δ = 0.0) as the internal standard or to the solvent
signals CDCl3 (δ = 7.26 ppm), CD3OD (δ = 3.31 or δ = 4.87) and C6D6 (δ = 7.16 ppm) and for
13C relative to CDCl3 (δ = 77.16), CD3OD (δ = 49.00) and C6D6 (δ = 128.06 ppm). The
13
13
multiplicity of the C NMR signals concerning the C-1H coupling was determined by the
HSQC method. Chemical shifts (in ppm) and coupling constants (in Hz) were obtained by
first-order analysis; assignments were derived from COSY and H/C correlation spectra.
Infrared (IR) spectra were measured with a Nicolet 6700 FT-IR spectrometer and reported in
wavenumber (cm-1). High-resolution mass spectra (HRMS) were recorded on a micrOTOF-Q
II quadrupole-time of flight hybrid mass spectrometer (Bruker Daltonics). Optical rotations
were determined using a P-2000 Jasco polarimeter. Melting points were recorded on a Kofler
hot block and are uncorrected. Microwave reactions were carried out on a focused microwave
system. The contents of the vessel were cooled rapidly using a stream of compressed air.
All commercial reagents were purchased from suppliers such as Sigma-Aldrich or Acros
Organics. Solvents were dried and purified before use according to standard procedures.
4.1.2. (S)-2-(Benzyloxy)-2-{(S)-1′,4′-dioxaspiro[4.5]decan-2′-yl}ethan-1-ol 2421
A solution of the known derivative 2320 (7.45 g, 35.1 mmol) in dry toluene (45 mL) was
successively treated with cyclohexanone (36 mL, 0.35 mol) and p-TsOH (0.67 g, 3.51 mmol).
After being stirred for 1 h at room temperature, the mixture was poured into a saturated
NH4Cl solution (45 mL) and extracted with EtOAc (2 × 70 mL). The combined organic layers
were dried over Na2SO4, concentrated, and the residue was subjected to flash chromatography
16

on silica gel (n-hexane/EtOAc, 3:1) to afford 8.70 g (85%) of compound 24 as a colourless
oil; [α]D21 –19.1 (c 1.16, CHCl3). IR (neat) υ 3439, 2932, 2860, 1449, 1365, 1280, 1163, 1094,
1067, 1027 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.37−1.44 (m, 2H, CH2), 1.55−1.69 (m, 8H,
4 × CH2), 2.27 (t, J = 5.4 Hz, 1H, OH), 3.54–3.61 (m, 2H, H-1, H-2), 3.67−3.74 (m, 1H, H-1),
3.77−3.82 (m, 1H, H-3'), 4.00 (dd, J = 6.6, 8.2 Hz, 1H, H-3'), 4.26–4.32 (m, 1H, H-2'ꞌ), 4.70
(d, J = 11.8 Hz, 1H, OCH2Ph), 4.79 (d, J = 11.8 Hz, 1H, OCH2Ph), 7.25−7.38 (m, 5H, Ph);
13C NMR (100 MHz, CDCl3) δ 24.0 (CH2), 24.2 (CH2), 25.3 (CH2), 34.9 (CH2), 36.2 (CH2),
61.9 (C-1), 65.4 (C-3'), 72.9 (OCH2Ph), 76.5 (C-2'), 79.5 (C-2), 110.2 (Cq), 128.0 (3 × CHPh),
128.6 (2 × CHPh), 138.4 (Ci). ESI-HRMS: m/z calcd for C17H25O4 [M + H]+ 293.1747, found
293.1759.
4.1.3. (S)-1-{(S)-1′,4′-Dioxaspiro[4.5]decan-2′-yl}ethane-1,2-diol 25
To a solution of 24 (8.60 g, 29.4 mmol) in dry EtOH (230 mL) were added a mixture of
catalysts 10% Pd/C/20% Pd(OH)2/C (1:1, 2.00 g). The resulting suspension was degassed
three times and was stirred for 3 h at room temperature under an atmosphere of hydrogen.
After this period, the mixture was filtered through a small pad of Celite, concentrated, and the
crude product was chromatographed on silica gel (EtOAc) to provide 5.50 g (92%) of
21
compound 25 as white crystals; mp 33−35 °C; [α]D +7.3 (c 0.18, CHCl3). IR (neat) υ 3408,
3363, 2934, 2852, 1445, 1366, 1285, 1092, 1066, 1028, 1012, 928 cm-1; 1H NMR (400 MHz,
CDCl3) δ 1.32–1.47 (m, 2H, CH2), 1.52–1.72 (m, 8H, 4 × CH2), 2.24−2.34 (m, 1H, OH),
2.57–2.64 (m, 1H, OH), 3.58–3.75 (m, 3H, H-1, 2 × H-2), 3.72–3.80 (m, 1H, H-3), 4.02–4.07
(m, 1H, H-3'), 4.15–4.21 (m, 1H, H-2'); 13C NMR (100 MHz, CDCl3) δ 23.9 (CH2), 24.2
(CH2), 25.2 (CH2), 34.9 (CH2), 36.3 (CH2), 64.5 (C-2), 65.7 (C-3'), 71.5 (C-1), 76.5 (C-2'),
110.4 (Cq). ESI-HRMS: m/z calcd for C10H18NaO4 [M + Na]+ 225.1097, found 225.1101.
4.1.4. Ethyl (R,E)-3-(1′,4′-dioxaspiro[4.5]decan-2′-yl)acrylate (E)-2622 and ethyl (R,Z)-3-
(1′,4′-dioxaspiro[4.5]decan-2′-yl)acrylate (Z)-26
Compound 25 (5.50 g, 27.2 mmol), which was dissolved in CH2Cl2 (150 mL), was treated
with a solution of NaIO4 (6.74 g, 31.5 mmol) in water (60 mL), and the resulting mixture was
stirred for 40 min at room temperature. The layers were separated and the aqueous one was
washed with further portions of CH2Cl2 (3 × 50 mL). The combined organic extracts were
dried over Na2SO4 and concentrated. The crude aldehyde30 (4.63 g) was used in subsequent
reaction without further purification.
17

Horner-Wadsworth-Emmons reaction: A suspension of NaH (1.40 g, 35.0 mmol, ~60%
dispersion in mineral oil) in dry THF (45 mL) was treated with (EtO)2P(O)CH2CO2Et (3.20
mL, 16.0 mmol) at 0 °C. After being stirred for 25 min at 0 °C, a solution of the crude
aldehyde (2.20 g, 12.90 mmol) in dry THF (12 mL) was added and stirring was continued for
20 min at the same temperature. The reaction was then quenched with a saturated NH4Cl
solution (30 mL) and the aqueous layer was washed with EtOAc (2 × 40 mL). The combined
organic extracts were dried over Na2SO4, the solvent was evaporated, and the residue was
purified by flash chromatography on silica gel (n-hexane/EtOAc, 15:1) to give 2.76 g (89%)
of compound (E)-26 as a colourless oil.
Wittig reaction: Ph3P=CHCO2Et (6.40 g, 18.40 mmol) was added to a solution of the crude
aldehyde (2.40 g, 14.10 mmol) in benzene (45 mL). After being stirred and refluxed for 25
min, the mixture was allowed to cool to room temperature, the solvent was evaporated, and
the residue was chromatographed on silica gel (n-hexane/EtOAc, 15:1) to afford 1.90 g (56%,
>97:3 dr) of (Z)-26 and 1.22 g (36%, >97:3 dr) of (E)-26 as colourless oils.
21
22
Ester (E)-26: Rf = 0.28 (n-hexane/EtOAc, 15:1); [α]D –31.9 (c 0.34, CHCl3); lit.22 [α]D
+31.2 (c 1.2, CHCl3); lit.31 [α]D30 +32.5 (c 0.7, CHCl3) for ent-(E)-26. IR (neat) υ 2934, 2862,
1717, 1661, 1448, 1301, 1261, 1161 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.29 (t, J = 7.1 Hz,
3H, CH3), 1.38–1.45 (m, 2H, CH2), 1.56–1.69 (m, 8H, 4 × CH2), 3.67 (t, J = 7.6 Hz, 1H, H-
3'), 4.15–4.24 (m, 3H, OCH2, H-3'), 4.63–4.69 (m, 1H, H-2'), 6.11 (dd, J = 0.9, 15.6 Hz, 1H,
13
H-2), 6.88 (dd, J = 5.6, 15.6 Hz, 1H, H-3); C NMR (100 MHz, CDCl3) δ 14.4 (CH3), 24.0
(CH2), 24.1 (CH2), 25.2 (CH2), 35.4 (CH2), 36.2 (CH2), 60.7 (OCH2), 68.6 (C-3'), 74.8 (C-2'),
111.0 (Cq), 122.5 (C-2), 145.1 (C-3), 166.2 (C=O). ESI-HRMS: m/z calcd for C13H20NaO4 [M
+ Na]+ 263.1254, found 263.1263.
22
27
Ester (Z)-26: Rf = 0.48 (n-hexane/EtOAc, 15:1); [α]D ‒77.0 (c 0.28, CHCl3); lit.32 [α]D
+84.6 (c 1.05, CHCl3) for ent-(Z)-26. IR (neat) υ 2981, 2934, 2862, 1715, 1643, 1448, 1367,
1278, 1161 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.29 (t, J = 7.1 Hz, 3H, CH3), 1.38–1.45 (m,
2H, CH2), 1.56–1.69 (m, 8H, 4 × CH2), 3.59–3.64 (m, 1H, H-3'), 4.17 (q, J = 7.1 Hz, 2H,
OCH2), 4.37 (t, J = 7.6 Hz, 1H, H-3'), 5.47–5.53 (m, 1H, H-2'), 5.83 (dd, J = 0.9, 11.7 Hz, 1H,
13
H-2), 6.36 (dd, J = 6.7, 11.7 Hz, 1H, H-3); C NMR (100 MHz, CDCl3) δ 14.3 (CH3), 23.9
(CH2), 24.0 (CH2), 25.2 (CH2), 35.0 (CH2), 36.3 (CH2), 60.5 (OCH2), 69.1 (C-3'), 73.2 (C-2'),
110.5 (Cq), 120.7 (C-2), 149.7 (C-3), 165.7 (C=O). ESI-HRMS: m/z calcd for C13H20NaO4 [M
+ Na]+ 263.1254, found 263.1266.
4.1.5. (R,E)-3-(1′,4′-Dioxaspiro[4.5]decan-2′-yl)prop-2-en-1-ol (E)-27
18

DIBAl-H (40.5 mL, 48.6 mmol, ~1.2 M solution in toluene) was added dropwise to a solution
of (E)-26 (3.90 g, 16.2 mmol) in dry CH2Cl2 (100 mL) that had been pre-cooled to −50 °C.
After being stirred for 20 min at −50 °C, the reaction was quenched by slow addition of
MeOH (9.0 mL). The whole mixture was then poured into a 30% K/Na tartrate solution (200
mL) and vigorous stirring was continued for 2 h at room temperature. Then the layers were
separated and the aqueous one was washed with CH2Cl2 (2 × 70 mL). The combined organic
extracts were dried over Na2SO4, concentrated, and the residue was subjected to flash
chromatography on silica gel (n-hexane/EtOAc, 2:1) to give 3.00 g (93%) of compound (E)-
27 as a colourless oil; [α]D22 –11.0 (c 0.54, CHCl3). IR (neat) υ 3467, 2933, 2860, 1716, 1664,
1
1448, 1364, 1279, 1162, 1095, 968 cm-1; H NMR (400 MHz, CDCl3) δ 1.35–1.43 (m, 2H,
CH2), 1.53–1.67 (m, 8H, 4 × CH2), 3.56–3.61 (m, 1H, H-3'), 4.08 (dd, J = 6.5, 7.8 Hz, 1H, H-
3'), 4.13–4.18 (m, 2H, 2 × H-1), 4.49–4.56 (m, 1H, H-2'), 5.67–5.74 (m, 1H, H-3), 5.95 (dt, J
= 5.2, 15.5 Hz, 1H, H-2); 13C NMR (CDCl3, 100 MHz) δ 24.0 (CH2), 24.1 (CH2), 25.3 (CH2),
35.6 (CH2), 36.4 (CH2), 62.8 (C-1), 69.2 (C-3'), 76.2 (C-2'), 110.1 (Cq), 128.6 (C-3), 133.4
(C-2). ESI-HRMS: m/z calcd for C11H18NaO3 [M + Na]+ 221.1148, found 221.1148.
4.1.6. (R,Z)-3-(1′,4′-Dioxaspiro[4.5]decan-2′-yl)prop-2-en-1-ol (Z)-27
Using the same procedure as described for the construction of (E)-27, ester (Z)-26 (1.84 g,
7.66 mmol) was reduced with DIBAl-H (19.2 mL, 23.0 mmol, ~1.2 M solution in toluene) to
afford after chromatography on silica gel (n-hexane/EtOAc, 2:1) 1.40 g (92%) of (Z)-27 as a
22
colourless oil; [α]D +12.3 (c 0.44, CHCl3); IR (neat) υ 3426, 2933, 2861, 1462, 1366, 1278,
1
1162, 1097, 1024, 847 cm-1; H NMR (400 MHz, CDCl3) δ 1.36–1.44 (m, 2H, CH2), 1.55–
1.67 (m, 8H, 4 × CH2), 2.07 (br s, 1H, OH), 3.54–3.59 (m, 1H, H-3'), 4.09 (dd, J = 6.7, 7.5
Hz, 1H, H-3'), 4.14–4.23 (m, 1H, H-1), 4.26–4.34 (m, 1H, H-1), 4.81–4.88 (m, 1H, H-2'),
5.53–5.60 (m, 1H, H-3), 5.79–5.86 (m, 1H, H-2); 13C NMR (100 MHz, CDCl3) δ 24.0 (CH2),
24.1 (CH2), 25.2 (CH2), 35.6 (CH2), 36.4 (CH2), 58.8 (C-1), 69.3 (C-3'), 71.7 (C-2'), 110.3
(Cq), 129.9 (C-3), 133.1 (C-2). ESI-HRMS: m/z calcd for C11H18NaO3 [M + Na]+ 221.1148,
found 221.1155.
4.1.7. N-{(R)-1'-[(R)-1'',4''-Dioxaspiro[4.5]decan-2''-yl]allyl}-2,2,2-trichloroacetamide 28
and N-{(S)-1'-[(R)-1'',4''-dioxaspiro[4.5]decan-2''-yl]allyl}-2,2,2-trichloroacetamide 29
4.1.7.1. General procedure for the preparation of trichloroacetimidates (E)-16 and (Z)-16
19

To a solution of (E)-27 or (Z)-27 (0.12 g, 0.61 mmol) in dry CH2Cl2 (3.0 mL) that had been
pre-cooled to 0 °C were successively added DBU (9.0 ꢁL, 0.06 mmol) and CCl3CN (0.12 mL,
1.22 mmol). After stirring for 15 min at 0 °C, the mixture was filtered through a small pad of
Celite, and the solvent was evaporated. This procedure yielded the corresponding crude
imidates (E)-16 or (Z)-16, which were then submitted to the next reaction without further
purification.
4.1.7.2. Microwave-assisted thermal Overman rearrangement (general procedure)
The corresponding imidate (E)-16 or (Z)-16 (0.10 g, 0.29 mmol) was weighed in a 10-mL
microwave tube equipped with a magnetic stirbar. o-Xylene (2.0 mL) and solid K2CO3 (46
mg, 0.33 mmol) were successively added, the tube was closed with septum, and the mixture
was subjected to microwave irradiation (for the temperatures and reaction times, see Table 1).
After completion, the insoluble parts were removed by filtration, the filtrate was concentrated,
and the residue was chromatographed on silica gel (n-hexane/EtOAc, 9:1) to give
trichloroacetamides 28 and 29 as an inseparable mixture of diastereoisomers (for the isolated
combined yields, see Table 1).
4.1.7.3. Pd(II)-catalyzed Overman rearrangement (general procedure)
A solution of (E)-16 or (Z)-16 (0.17 g, 0.50 mmol) in dry toluene (2.0 mL) was successively
treated with PdCl2(MeCN)2 (10 mg, 0.04 mmol) and p-benzoquinone (108 mg, 1.00 mmol).
The resulting mixture was stirred and heated either for 40 °C or 70 °C (for the reaction times,
see Table 1). After being cooled to room temperature, the mixture was filtered through a small
pad of Celite, concentrated, and the residue was chromatographed on silica gel (n-
hexane/EtOAc, 9:1) to provide the corresponding amides 28 and 29 in the combined yield of
40%, but only in the case of (E)-16 (see Table 1).
1
Spectral data for both diastereoisomeric amides 28 and 29: H NMR (400 MHz, CDCl3) δ
1.32−1.47 (m, 2.66H, CH2-28, CH2-29), 1.50−1.69 (m, 10.72H, 4 × CH2-28, 4 × CH2-29),
3.68 (dd, J = 6.5, 8.6 Hz, 1H, H-3''-29), 3.82 (dd, J = 5.2, 8.9 Hz, 1H, H-3''-28), 4.04−4.10 (m,
1.33H, H-3''-28, H-3''-29), 4.27−4.34 (m, 1.33H, H-2''-28, H-2''-29), 4.44−4.51 (m, 1.33H, H-
1'-28, H-1'-29), 5.27−5.38 (m, 2.66H, H-3'cis-28, H-3'cis-29, H-3'trans-28, H-3'trans-29),
5.80−5.92 (m, 1.33H, H-2'-28, H-2'-29), 7.01−7.15 (m, 1.33H, NH-28, NH-29); 13C NMR
(100 MHz, CDCl3) δ 23.8 (CH2-28, CH2-29), 24.1 (CH2-28, CH2-29), 25.2 (CH2-28, CH2-29),
34.2 (CH2-28), 34.5 (CH2-29), 35.9 (CH2-28), 36.2 (CH2-29), 54.3 (C-1'-29), 56.0 (C-1'-28),
65.3 (C-3''-28), 66.2 (C-3''-29), 75.9 (C-2''-28), 76.3 (C-2''-29), 92.7 (CCl3-28, CCl3-29),
20

110.7 (Cq-29), 110.9 (Cq-28), 117.9 (C-3'-29), 119.3 (C-3'-28), 131.9 (C-2'-28), 134.1 (C-2'-
29), 161.5 (C=O-28), 162.0 (C=O-29).
4.1.8. (R,E)-2-(3'-Thiocyanatoprop-1′-en-1'-yl)-1,4-dioxaspiro[4.5]decane (E)-17
To a solution of (E)-27 (2.70 g, 13.6 mmol) in dry CH2Cl2 (115 mL) were successively added
Et3N (3.82 mL, 27.2 mmol) and MsCl (2.10 mL, 27.2 mmol) at 0 °C. After being stirred for
20 min at the same temperature, the solvent was evaporated, and the residue was treated with
Et2O (40 mL). The insoluble parts were removed by filtration, the filtrate was concentrated,
and the obtained mesylate was subjected to the subsequent reaction without further
purification.
KSCN (2.00 g, 20.6 mmol) was added to a solution of the crude mesylate (3.76 g, 13.6 mmol)
in dry acetonitrile (120 mL) and the resulting mixture was stirred for 2 h at room temperature.
After completion of the reaction, the solvent was evaporated, and to the crude product was
added Et2O (40 mL). The generated solid material was filtered off, the solution was
concentrated, and the residue was chromatographed on silica gel (n-hexane/EtOAc, 7:1) to
21
furnish 2.67 g (82%) of thiocyanate (E)-17 as a colourless oil; [α]D –47.2 (c 2.94, CHCl3).
IR (neat) υ 2933, 2860, 2154, 1462, 1448, 1364, 1279, 1231, 1161, 1039 cm-1; 1H NMR (400
MHz, CDCl3) δ 1.34–1.42 (m, 2H, CH2), 1.52–1.65 (m, 8H, 4 × CH2), 3.48–3.59 (m, 2H, 2 ×
H-3ꞌ), 3.59–3.64 (m, 1H, H-3), 4.12 (dd, J = 6.9, 7.7 Hz, 1H, H-3), 4.50–4.57 (m, 1H, H-2),
13
5.77–5.92 (m, 2H, H-1', H-2'); C NMR (100 MHz, CDCl3) δ 23.9 (CH2), 24.0 (CH2), 25.2
(CH2), 35.4 (CH2), 35.5 (C-3'), 36.2 (CH2), 69.2 (C-3), 75.3 (C-2), 110.5 (Cq), 111.6 (SCN),
125.2 (C-2'), 135.7 (C-1'). ESI-HRMS: m/z calcd for C12H17NNaO2S [M + Na]+ 262.0872,
found 262.0879.
4.1.9. (R,Z)-2-(3'-Thiocyanatoprop-1′-en-1'-yl)-1,4-dioxaspiro[4.5]decane (Z)-17
By the same procedure as employed for the conversion of (E)-27 into (E)-17, compound (Z)-
27 (1.00 g, 5.04 mmol) was transformed to thiocyanate (Z)-17 (1.09 g, 90%, colourless oil, n-
hexane/EtOAc, 7:1); [α]D21 +132.7 (c 1.16, CHCl3). IR (neat) υ 2933, 2859, 2154, 1462, 1448,
1
1367, 1278, 1231, 1161, 1039 cm-1; H NMR (400 MHz, CDCl3) δ 1.35–1.44 (m, 2H, CH2),
1.54–1.67 (m, 8H, 4 × CH2), 3.54–3.65 (m, 2H, H-3, H-3ꞌ), 3.84–3.94 (m, 1H, H-3'), 4.15 (dd,
13
J = 6.2, 8.3 Hz, 1H, H-3), 4.75–4.82 (m, 1H, H-2), 5.72–5.80 (m, 2H, H-1', H-2'); C NMR
(100 MHz, CDCl3): δ 24.0 (CH2), 24.1 (CH2), 25.2 (CH2), 31.0 (C-3'), 35.5 (CH2), 36.4
(CH2), 69.5 (C-3), 71.3 (C-2), 110.7 (Cq), 111.7 (SCN), 125.6 (C-2'), 134.3 (C-1'). ESI-
HRMS: m/z calcd for C12H17NNaO2S [M + Na]+ 262.0872, found 262.0880.
21

4.1.10. (R)-2-[(R)-1′-Isothiocyanatoallyl]-1,4-dioxaspiro[4.5]decane 30 and (R)-2-[(S)-1′-
isothiocyanatoallyl]-1,4-dioxaspiro[4.5]decane 31
4.1.10.1. Conventional aza-Claisen rearrangement (general procedure)
A solution of the corresponding thiocyanate (E)-17 or (Z)-17 (0.10 g, 0.42 mmol) in n-heptane
(2 mL) was stirred and heated under a nitrogen atmosphere (for the temperatures and reaction
times, see Table 1). After completion of the reaction, the mixture was allowed to cool to room
temperature, and the solvent was evaporated. Chromatography of the residue on silica gel (n-
hexane/EtOAc, 19:1) delivered isothiocyanates 30 and 31 as a separable mixture of
diastereoisomers (for the isolated combined yields, see Table 2).
4.1.10.2. Microwave-assisted synthesis (general procedure)
Thiocyanate (E)-16 or (Z)-16 (0.10 g, 0.42 mmol) was weighed in a 10-mL tube equipped
with a magnetic stirbar. Then n-heptane (2 mL) was added, and the mixture was subjected to
microwave irradiation (for the temperatures and reaction times, see Table 2). After
completion of the reaction, the solvent was removed under reduced pressure, and the residue
was chromatographed on silica gel (n-hexane/EtOAc, 19:1) to afford a mixture of 30 and 31
(for the combined yields, see Table 2).
Requiring greater amounts of the rearranged products 30 and 31, they were prepared on a
multigram scale by the conventional method at 70 °C starting from (E)-17.
21
Isothiocyanate 30: colourless oil, Rf = 0.50 (n-hexane/EtOAc, 19:1); [α]D +29.5 (c 0.42,
1
CHCl3). IR (neat) υ 2934, 2860, 2049, 2034, 1645, 1500, 1448, 1332, 1279, 1160 cm -1; H
NMR (400 MHz, CDCl3) δ 1.36–1.45 (m, 2H, CH2), 1.52–1.72 (m, 8H, 4 × CH2), 3.91 (dd, J
= 5.2, 8.7 Hz, 1H, H-3), 4.05 (dd, J = 6.3, 8.7 Hz, 1H, H-3), 4.10–4.15 (m, 1H, H-2), 4.38–
4.43 (m, 1H, H-1'), 5.33 (dd, J = 0.5, 10.3 Hz, 1H, H-3'cis), 5.45 (dd, J = 0.5, 17.0 Hz, 1H, H-
13
3'trans), 5.81 (ddd, 1H, J = 5.0, 10.3, 17.0 Hz, 1H, H-2'); C NMR (100 MHz, CDCl3) δ 23.8
(CH2), 24.0 (CH2), 25.1 (CH2), 34.7 (CH2), 36.3 (CH2), 61.7 (C-1'), 65.6 (C-3), 76.9 (C-2),
111.3 (Cq), 118.4 (C-3'), 131.7 (C-2'), 135.1 (NCS). ESI-HRMS: m/z calcd for
C12H17NNaO2S [M + Na]+ 262.0872, found 262.0880.
21
Isothiocyanate 31: colourless oil, Rf = 0.36 (n-hexane/EtOAc, 19:1); [α]D –78.6 (c 0.46,
1
CHCl3). IR (neat) υ 2934, 2860, 2048, 2033, 1644, 1448, 1365, 1280, 1161 cm-1; H NMR
(400 MHz, CDCl3) δ 1.35–1.44 (m, 2H, CH2), 1.51–1.72 (m, 8H, 4 × CH2), 3.80 (dd, J = 5.3,
8.7 Hz, 1H, H-3), 4.04 (dd, J = 6.3, 8.7 Hz, 1H, H-3), 4.16–4.21 (m, 1H, H-2), 4.22–4.26 (m,
22

1H, H-1'), 5.35 (d, J = 10.3 Hz, 1H, H-3'cis), 5.44 (d, J = 17.0 Hz, 1H, H-3ꞌtrans), 5.73–5.87 (m,
13
1H, H-2'); C NMR (100 MHz, CDCl3) δ 23.9 (CH2), 24.1 (CH2), 25.2 (CH2), 34.7 (CH2),
36.3 (CH2), 62.3 (C-1'), 65.7 (C-3), 77.0 (C-2), 111.3 (Cq), 119.4 (C-3'), 131.6 (C-2'), 135.6
(NCS). ESI-HRMS: m/z calcd for C12H17NNaO2S [M + Na]+ 262.0872, found 262.0878.
4.1.11.
(4R,5R)-5-(Hydroxymethyl)-4-vinyloxazolidin-2-one
3315
and
(4S,5R)-5-
(hydroxymethyl)-4-vinyloxazolidin-2-one 3415
To a solution of the mixture of amides 28 and 29 (0.16 g, 0.47 mmol, dr = 75:25) in MeOH (8
mL) was added p-TsOH (27 mg, 0.14 mmol) at room temperature. After being stirred and
heated for 6 h at 65 °C, the mixture was quenched by neutralization with Et3N, and the
solvent was evaporated. Chromatography of the residue on silica gel (n-hexane/EtOAc, 1:2)
afforded 0.12 g (95%) of diols 32 as an inseparable mixture of diastereoisomers.
1
The spectroscopic data for the corresponding mixture of diols 32: H NMR (400 MHz,
CD3OD) δ 3.53−3.56 (m, 0.66H, 2 × H-5'min), 3.62–3.65 (m, 2H, 2 × H-5'maj), 3.73–3.77 (m,
1H, H-4'maj), 3.82 (td, 0.33H, J = 4.1, 6.1 Hz, 1H, H-4'min), 4.46–4.50 (m, 1H, H-3'maj), 4.50–
4.51 (m, 0.33H, H-3'min), 5.23–5.25 (m, 0.66H, 2 × H-1'min), 5.25–5.32 (m, 2H, 2 × H-1'maj),
13
5.90–6.01 (m, 1.33H, H-2'maj, H-2'min); C NMR (100 MHz, CD3OD) δ 57.0 (C-3'min), 58.1
(C-3'maj), 64.2 (C-5'min), 64.3 (C-5'maj), 73.2 (C-4'maj), 73.4 (C-4'min), 94.0 (CCl3maj, CCl3min),
117.4 (C-1'min), 117.9 (C-1'maj), 134.6 (C-2'maj), 135.6 (C-2'min), 163.4 (C=Omaj, C=Omin).
DBU (21 ꢁL, 0.14 mmol) was added to a solution of stereoisomers 32 (0.12 g, 0.46 mmol) in
dry CH2Cl2 (4.0 mL) at 0 °C and the resulting mixture was stirred for 21 h at room
temperature. After completion of the reaction, the mixture was filtered through a small pad of
Celite and concentrated. Chromatography of the residue on silica gel (EtOAc) gave 39 mg
(60%) of compound 33 and 13 mg (20%) of 34. The obtained oxazolidin-2-ones had
spectroscopic data and optical rotation values in very good agreement with those reported in
the literature for the same derivatives.
22
Diastereoisomer 33: white crystals; mp 102−104 °C; [α]D –8.1 (c 0.40, MeOH); lit.15 mp
26
102−105 °C; [α]D –7.0 (c 0.37, MeOH). IR (neat) υ 3438, 3239, 2978, 2953, 2931, 1742,
1
1690, 1408, 1314, 1246, 1117 cm-1; H NMR (400 MHz, CD3OD) δ 3.65−3.68 (m, 2H,
CH2OH), 4.44–4.49 (m, 1H, H-4), 4.71 (ddd, J = 4.9, 6.0, 8.6 Hz, 1H, H-5), 5.29 (dt, J = 1.1,
10.3 Hz, 1H, Hcis-CH2), 5.35 (dt, J = 1.1, 17.3 Hz, 1H, Htrans-CH2), 5.94 (ddd, J = 7.1, 10.3,
17.3 Hz, 1H, CHvinyl); 13C NMR (100 MHz, CD3OD) δ 58.1 (C-4), 61.7 (CH2OH), 81.4 (C-5),
23

119.2 (CH2vinyl), 134.6 (CHvinyl), 161.5 (C=O). ESI-HRMS: m/z calcd for C6H9NNaO3 [M +
Na]+ 166.0475, found 166.0483.
22
26
Diastereoisomer 34: colourless oil; [α]D –85.1 (c 0.15, MeOH); lit.15 [α]D –87.5 (c 0.16,
1
MeOH). IR (neat) υ 3278, 2925, 1720, 1647, 1390, 1281, 1224, 1097 cm-1; H NMR (400
MHz, CD3OD) δ 3.64 (dd, J = 4.1, 12.6 Hz, 1H, H-CH2OH), 3.78 (dd, J = 3.2, 12.6 Hz, 1H,
H-CH2OH), 4.20–4.28 (m, 2H, H-4, H-5), 5.24 (dt, J = 0.9, 10.3 Hz, 1H, Hcis-CH2), 5.33 (dt, J
= 0.9, 17.1 Hz, 1H, Htrans-CH2), 5.90 (ddd, J = 6.7, 10.3, 17.1 Hz, 1H, CHvinyl); 13C NMR (100
MHz, CD3OD) δ 57.9 (C-4), 62.3 (CH2OH), 84.0 (C-5), 118.2 (CH2vinyl), 137.9 (CHvinyl),
161.4 (C=O). ESI-HRMS: m/z calcd for C6H9NNaO3 [M + Na]+ 166.0475, found 166.0476.
4.1.12. (4S,5R)-5-(Hydroxymethyl)-4-vinyloxazolidine-2-thione 3515
Compound 35 (32 mg, 96%) was prepared according to the same procedure as described in
the literature15 starting from the minor isothiocyanate 31 (50 mg, 0.21 mmol). Spectroscopic
data, optical rotation and melting point of the obtained derivative 35 were in very good accord
with those reported in the literature for the same compound; mp 130−132 °C; [α]D21 −158.3 (c
26
0.60, CHCl3); lit.15 mp 130−133 °C; [α]D −144.6 (c 0.48, MeOH). IR (neat) υ 3330, 3180,
1
2928, 1522, 1380, 1257, 1177 cm-1; H NMR (400 MHz, CD3OD) δ 3.67 (dd, J = 4.1, 12.8
Hz, 1H, H-CH2OH), 3.82 (dd, J = 4.1, 12.8 Hz, 1H, H-CH2OH), 4.40 (t, J = 7.1 Hz, 1H, H-4),
4.50−4.55 (m, 1H, H-5), 5.29 (d, J = 10.2 Hz, 1H, Hcis-CH2), 5.35 (d, J = 17.1 Hz, 1H, Htrans
-
13
CH2), 5.89 (ddd, J = 7.1, 10.2, 17.1 Hz, 1H, CHvinyl); C NMR (100 MHz, CD3OD) δ 61.6
(C-4), 62.0 (CH2OH), 89.5 (C-5), 119.3 (CH2vinyl), 136.4 (CHvinyl), 190.6 (C=S). ESI-HRMS:
m/z calcd for C6H9NNaO2S [M + Na]+ 182.0246, found 182.0248.
4.1.13. tert-Butyl {(R)-1-[(R)-1′,4′-dioxaspiro[4.5]decan-2′-yl]allyl}carbamate 19
TBTO (6.8 mL, 13.3 mmol) was added to a solution of 30 (1.60 g, 6.68 mmol) in dry toluene
(37 mL) at room temperature. After being stirred and heated for 3 h at 60 °C, the mixture was
allowed to cool to room temperature, and the solvent was evaporated. Chromatography of the
residue on silica gel (EtOAc/MeOH/Et3N, 80:20:1) afforded 1.25 g (95%) of the crude amine
as a colourless oil, which was immediately submitted to the next reaction without spectral
characterisation.
A solution of the obtained amine (1.25 g, 6.34 mmol) in dry CH2Cl2 (20 mL) was
successively treated with Et3N (1.0 mL, 7.12 mmol) and Boc2O (2.10 g, 9.62 mmol). After
stirring at room temperature overnight, the mixture was poured into a saturated NH4Cl
solution (20 mL) and was extracted with CH2Cl2 (2 × 20 mL). The combined organic layers
were dried over Na2SO4, concentrated, and the residue was subjected to flash chromatography
24

on silica gel (n-hexane/EtOAc, 11:1) to give 1.55 g (82%) of 19 as white crystals; mp 66−68
°C; [α]D21 +41.6 (c 0.44, CHCl3). IR (neat) υ 3294, 2981, 2942, 2860, 1680, 1532, 1366, 1272,
1
1161 cm-1; H NMR (400 MHz, CDCl3) δ 1.31–1.46 (m, 11H, 3 × CH3, CH2), 1.50–1.67 (m,
8H, 4 × CH2), 3.77 (dd, J = 5.7, 8.5 Hz, 1H, H-3'), 4.01 (dd, J = 6.9, 8.5 Hz, 1H, H-3'), 4.10–
4.23 (m, 2H, H-1, H-2'), 4.80 (br s, 1H, NH), 5.22 (d, J = 10.2 Hz, 1H, H-3cis), 5.25 (d, J =
13
17.3 Hz, 1H, H-3trans), 5.80−5.90 (m, 1H, H-2); C NMR (100 MHz, CDCl3) δ 23.9 (CH2),
24.1 (CH2), 25.3 (CH2), 28.5 (3 × CH3), 34.7 (CH2), 36.1 (CH2), 55.2 (C-1), 65.9 (C-3'), 77.2
(C-2'), 79.8 (Cq), 110.4 (Cq), 117.3 (C-3), 134.6 (C-2), 155.5 (C=O). ESI-HRMS: m/z calcd
for C16H27NNaO4 [M + Na]+ 320.1832, found 320.1845.
4.1.14. Ethyl (R,E)-4-[(tert-butoxycarbonyl)amino]-4-[(R)-1′,4′-dioxaspiro[4.5]decan-2′-
yl]but-2-enoate 36
Ozone was introduced to a solution of 19 (1.40 g, 4.71 mmol) in dry EtOH (40 mL) at –78 °C
for 45 min. This resulted in the formation of a slightly blue solution. After excess ozone was
purged with dry nitrogen, a solution of Ph3P (1.24 g, 4.71 mmol) in dry CH2Cl2 (20 mL) was
added at ‒78 °C, and the resulting mixture was stirred for another hour at room temperature.
Then, the solvent was evaporated and the crude aldehyde was submitted to the subsequent
reaction without further purification.
A suspension of NaH (0.50 g, 12.6 mmol, ~60% dispersion in mineral oil) in dry THF (9.5
mL) was treated with (EtO)2P(O)CH2CO2Et (1.18 mL, 5.89 mmol) at 0 °C. After stirring for
25 min at 0 °C, a solution of the obtained aldehyde (1.41 g, 4.71 mmol) in dry THF (6.5 mL)
was added and stirring was continued for 30 min at the same temperature. The reaction was
quenched with a saturated NH4Cl solution (15 mL). The aqueous phase was separated and
was washed with EtOAc (2 × 20 mL). The combined organic extracts were dried over
Na2SO4, the solvent was evaporated, and the residue was chromatographed on silica gel (n-
21
hexane/EtOAc, 7:1) to provide 1.36 g (78%) of compound 36 as a colourless oil; [α]D +9.3
(c 0.44, CHCl3). IR (neat) υ 3342, 2978, 2934, 2862, 1701, 1659, 1517, 1449, 1366, 1249,
1
1158 cm-1; H NMR (400 MHz, CDCl3) δ 1.29 (t, J = 7.1 Hz, 3H, CH3), 1.34–1.42 (m, 2H,
CH2), 1.45 (s, 9H, 3 × CH3), 1.51–1.65 (m, 8H, 4 × CH2), 3.79 (dd, J = 5.3, 8.7 Hz, 1H, H-3'),
4.04 (dd, J = 6.9, 8.7 Hz, 1H, H-3'), 4.13–4.24 (m, 3H, OCH2, H-2'), 4.33−4.43 (m, 1H, H-4),
4.86 (d, J = 7.8 Hz, 1H, NH), 6.00 (d, J = 15.8 Hz, 1H, H-2), 6.93 (dd, J = 5.5, 15.8 Hz, 1H,
H-3); 13C NMR (100 MHz, CDCl3) δ 14.3 (CH3), 23.9 (CH2), 24.1 (CH2), 25.2 (CH2), 28.5 (3
× CH3), 34.6 (CH2), 36.1 (CH2), 54.1 (C-4), 60.7 (OCH2), 65.9 (C-3'), 77.4 (C-2'), 80.3 (Cq),
25

110.9 (Cq), 123.1 (C-3), 144.2 (C-2), 155.2 (C=O), 166.1 (C=O). ESI-HRMS: m/z calcd for
C19H31NNaO6 [M + Na]+ 392.2044, found 392.2046.
4.1.15.
tert-Butyl
{(R,E)-4-hydroxy-1-[(R)-1′,4′-dioxaspiro[4.5]decan-2′-yl]but-2-en-1-
yl}carbamate 37
A solution of 36 (1.36 g, 3.68 mmol) in dry CH2Cl2 (17 mL) was treated with BF3.OEt2 (0.57
mL, 4.60 mmol) at −78 °C, and the resulting solution was stirred for 25 min at the same
temperature. Then DIBAl-H (16.8 mL, 20.2 mmol, ~1.2 M solution in toluene) was added
dropwise at ‒78 °C and stirring was continued for another 30 min. The reaction was quenched
by slow addition of MeOH (4.2 mL), and the whole mixture was then poured into a 30%
K/Na tartrate solution (85 mL). After stirring for 2 h at room temperature, the organic layer
was separated and the aqueous one was washed with CH2Cl2 (2 × 35 mL). The combined
organic extracts were dried over Na2SO4, stripped solvent, and the residue was
chromatographed on silica gel (n-hexane/EtOAc, 1:1) to give 1.10 g (91%) of compound 37
21
as a colourless oil; [α]D +12.3 (c 0.40, CHCl3). IR (neat) υ 3435, 3336, 3005, 2976, 2933,
2862, 1690, 1517, 1365, 1249, 1160, 1041 cm-1; 1H NMR (400 MHz, CDCl3) δ 1.29−1.47 (m,
11H, 3 × CH3, CH2), 1.49–1.66 (m, 8H, 4 × CH2), 1.75 (br s, 1H, OH), 3.76 (dd, J = 5.6, 8.5
Hz, 1H, H-3'), 4.00 (dd, J = 6.6, 8.5 Hz, 1H, H-3'), 4.09–4.25 (m, 4H, H-1, H-2', 2 × H-4),
4.83 (br s, 1H, NH), 5.72 (dd, J = 5.9, 15.5 Hz, 1H, H-2), 5.85 (dt, J = 5.1, 15.5 Hz, 1H, H-3);
13C NMR (100 MHz, CDCl3) δ 23.9 (CH2), 24.1 (CH2), 25.3 (CH2), 28.5 (3 × CH3), 34.6
(CH2), 36.1 (CH2), 54.3 (C-1), 63.1 (C-4), 66.0 (C-3'), 77.3 (C-2'), 79.9 (Cq), 110.5 (Cq),
127.7 (C-3), 132.2 (C-2), 155.4 (C=O). ESI-HRMS: m/z calcd for C17H29NNaO5 [M + Na]+
350.1938, found 350.1954.
4.1.16. tert-Butyl {(R,E)-1-[(R)-1′,4′-dioxaspiro[4.5]decan-2′-yl]-4-thiocyanatobut-2-en-1-
yl}carbamate 18
Using the same procedure as described for the transformation (E)-27 into (E)-17, compound
37 (1.10 g, 3.36 mmol) was converted into thiocyanate 18 (colourless oil, 1.08 g, 87%, (n-
hexane/EtOAc, 3:1); [α]D21 −18.5 (c 1.12, CHCl3). IR (neat) υ 3344, 2933, 2861, 2154, 1697,
1
1506, 1449, 1365, 1233, 1160 cm-1; H NMR (400 MHz, CDCl3) δ 1.33–1.46 (m, 11H, 3 ×
CH3, CH2), 1.49–1.65 (m, 8H, 4 × CH2), 3.51−3.62 (m, 2H, 2 × H-4), 3.75−3.80 (m, 1H, H-
3'), 3.99–4.04 (m, 1H, H-3'), 4.10−4.17 (m, 1H, H-2'), 4.19−4.30 (m, 1H, H-1), 4.87 (br d, J =
13
6.9 Hz, 1H, NH), 5.75−5.88 (m, 2H, H-2, H-3); C NMR (100 MHz, CDCl3) δ 23.8 (CH2),
24.1 (CH2), 25.2 (CH2), 28.4 (3 × CH3), 34.5 (CH2), 35.9 (C-4), 36.1 (CH2), 54.1 (C-1), 65.8
(C-3'), 76.8 (C-2'), 80.1 (Cq), 110.6 (Cq), 111.7 (SCN), 125.1 (C-2 or C-3), 134.2 (C-2 or C-
26

3), 155.3 (C=O). ESI-HRMS: m/z calcd for C18H28N2NaO4S [M + Na]+ 391.1662, found
391.1672.
4.1.17. tert-Butyl {(1R,2S)-2-isothiocyanato-1-[(R)-1′,4′-dioxaspiro[4.5]decan-2′-yl]but-3-en-
1-yl}carbamate
38
and
tert-butyl
{(1R,2R)-2-isothiocyanato-1-[(R)-1′,4′-
dioxaspiro[4.5]decan-2′-yl]but-3-en-1-yl}carbamate 39
4.1.17.1. Thermal aza-Claisen rearrangement (general procedure)
By the same procedure employed for the thermally driven aza-Claisen rearrangement of
thiocyanates (E/Z)-17, compound 18 (75 mg, 0.20 mmol) was converted into the
corresponding isothiocyanates 38 and 39 (n-hexane/EtOAc, 9:1, for the temperatures, reaction
times and combined yields, see Table 3).
4.17.2. Microwave-assisted synthesis (general procedure)
According to the same procedure described for the microwave-promoted [3,3]-sigmatropic
rearrangement of thiocyanates (E/Z)-17, compound 18 (75 mg, 0.20 mmol) was transformed
to rearranged products 38 and 39 (n-hexane/EtOAc, 9:1, for the temperatures, reaction times
and combined yields, see Table 3).
Requiring greater amounts of the rearranged products 38 and 39, they were prepared on a
multi-gram scale by the microwave-assisted synthesis at 70 °C starting from 18.
21
Isothiocyanate 38: colourless oil; Rf = 0.32 (n-hexane/EtOAc, 9:1); [α]D −101.6 (c 0.80,
CHCl3); IR (neat) υ 3338, 2927, 2853, 2054, 1705, 1499, 1449, 1248, 1158, 1096, 1041 cm-1;
1H NMR (400 MHz, C6D6) δ 1.15−1.27 (m, 2H, CH2), 1.35 (s, 9H, 3 × CH3), 1.43–1.60 (m,
8H, 4 × CH2), 3.60−3.67 (m, 1H, H-2'), 3.70−3.79 (m, 2H, 2 × H-3'), 3.83–3.90 (m, 1H, H-1),
4.18 (d, J = 10.0 Hz, 1H, NH), 4.61−4.65 (m, 1H, H-2), 4.89−4.94 (m, 1H, H-4cis), 5.15−5.22
(m, 1H, H-4trans), 5.48−5.58 (m, 1H, H-3); 13C NMR (100 MHz, C6D6) δ 24.1 (CH2), 24.4
(CH2), 25.5 (CH2), 28.3 (3 × CH3), 35.3 (CH2), 37.0 (CH2), 56.5 (C-1), 61.7 (C-2), 67.3 (C-
3'), 74.9 (C-2'), 79.8 (Cq), 110.9 (Cq), 117.2 (C-4), 133.2 (C-3), 137.3 (NCS), 155.4 (C=O).
ESI-HRMS: m/z calcd for C18H28N2NaO4S [M + Na]+ 391.1662, found 391.1671.
21
Isothiocyanate 39: colourless oil; Rf = 0.17 (n-hexane/EtOAc, 9:1); [α]D −22.0 (c 0.46,
CHCl3). IR (neat) υ 3338, 2934, 2861, 2051, 1693, 1503, 1449, 1278, 1234, 1159, 1095, 1039
1
cm-1; H NMR (400 MHz, C6D6, 70 °C) δ 1.18−1.29 (m, 2H, CH2), 1.44 (s, 9H, 3 × CH3),
1.45–1.55 (m, 8H, 4 × CH2), 3.48−3.55 (m, 1H, H-2'), 3.76 (dd, J = 6.1, 8.6 Hz, 1H, H-3'),
3.81 (dd, J = 6.0, 8.6 Hz, 1H, H-3'), 4.01–4.09 (m, 1H, H-1), 4.20 (d, J = 9.1 Hz, 1H, NH),
4.32−4.36 (m, 1H, H-2), 4.92 (dd, J = 0.7, 10.3 Hz, 1H, H-4cis), 5.20 (dd, J = 0.7, 17.0 Hz,
27

1H, H-4trans), 5.41−5.53 (m, 1H, H-3); 13C NMR (100 MHz, C6D6) δ 24.2 (CH2), 24.3 (CH2),
25.4 (CH2), 28.5 (3 × CH3), 35.4 (CH2), 36.7 (CH2), 56.9 (C-1), 60.7 (C-2), 67.3 (C-3'), 75.0
(C-2'), 80.2 (Cq), 110.8 (Cq), 119.0 (C-4), 131.1 (C-3), 136.7 (NCS), 155.3 (C=O). ESI-
HRMS: m/z calcd for C18H28N2NaO4S [M + Na]+ 391.1662, found 391.1670.
4.1.18.
Di-tert-butyl
{(1R,2S)-1-[(R)-1',4'-dioxaspiro[4.5]decan-2'-yl]but-3-ene-1,2-
diyl}dicarbamate 21
To a solution of 38 (0.55 g, 1.49 mmol) in dry toluene (8.2 mL) was added TBTO (1.52 mL,
2.98 mmol) at room temperature. The resulting mixture was stirred and heated for 3 h at 60
°C. After completion of the reaction, the solvent was evaporated, and the residue was
chromatographed on silica gel (EtOAc/0.5% Et3N) to afford 0.36 g (73%) of the
corresponding amine as a colourless oil, which was immediately subjected to the next reaction
without spectral characterization.
A solution of the obtained amine (0.36 g, 1.10 mmol) in dry CH2Cl2 (3.6 mL) was
successively treated with Et3N (0.18 mL, 1.26 mmol) and Boc2O (0.36 g, 1.65 mmol). After
being stirred for 16 h at room temperature, the mixture was poured into a saturated NH4Cl
solution (4 mL) and was extracted with CH2Cl2 (2 × 10 mL). The combine organic extracts
were dried over Na2SO4, stripped solvent, and the residue was purified by flash
chromatography on silica gel (n-hexane/EtOAc, 5:1) to give 0.42 g (89%) of compound 21 as
21
white crystals; mp 103−105 °C; [α]D −44.6 (c 1.04, CHCl3). IR (neat) υ 3368, 3024, 3005,
1
2970, 2932, 2862, 1737, 1725, 1692, 1518, 1449, 1230, 1160 cm-1; H NMR (400 MHz,
C6D6, 70 °C) δ 1.23−1.30 (m, 2H, CH2), 1.41 (s, 9H, 3 × CH3), 1.44 (s, 9H, 3 × CH3), 1.53–
1.69 (m, 8H, 4 × CH2), 3.85−4.02 (m, 4H, H-1, H-2', 2 × H-3'), 4.45−4.59 (m, 1H, H-2), 5.00
13
(d, J = 10.4 Hz, 1H, H-4cis), 5.16 (d, J = 17.2 Hz, 1H, H-4trans), 5.72−5.83 (m, 1H, H-3); C
NMR (100 MHz, C6D6, 70 °C) δ 24.3 (CH2), 24.5 (CH2), 25.7 (CH2), 28.5 (3 × CH3), 28.6 (3
× CH3), 35.6 (CH2), 36.9 (CH2), 55.8 (C-1 or C-2), 57.0 (C-1 or C-2), 67.3 (C-3'), 76.0 (C-2'),
79.3 (Cq), 79.5 (Cq), 110.5 (Cq), 116.2 (C-4), 136.6 (C-3), 156.0 (C=O), 156.1 (C=O). ESI-
HRMS: m/z calcd for C22H39N2O6 [M + H]+ 427.2803, found 427.2816.
4.1.19.
Di-tert-butyl
{(1R,2R)-1-[(R)-1',4'-dioxaspiro[4.5]decan-2'-yl]but-3-ene-1,2-
diyl}dicarbamate 22
By the same procedure as employed for the conversion of 38 to 21, compound 39 (0.16 g,
0.43 mmol) was transformed over two steps to derivative 22 (white crystals, 0.11 g, 60%, n-
21
hexane/EtOAc, 5:1); mp 196−197 °C; [α]D +18.4 (c 0.58, CHCl3). IR (neat) υ 3363, 3078,
28