Synthesis of conformationally-constrained thio(seleno)hydantoins and α-triazolyl lactones from d-arabinose as potential glycosidase inhibitors

Article abstract of DOI:10.1016/j.tet.2012.03.087

We have explored the rich structural diversity provided by an α-azido ester derived from d-arabinose as the source of sugar templates with reduced conformational flexibility. Using transient α-thioureido(selenoureido) esters we have prepared spiranic thio(seleno)hydantoins at the C-3 position of the sugar moiety. In this context, the first example of a stable spiranic α-lactam (or aziridinone) was isolated as a by-product in the hydrogenolysis of the starting α-azido ester. Furthermore, using copper(I)-catalyzed azido-alkyne cycloaddition (click chemistry), we have accessed bicyclic cis-fused α-triazolyl lactones fixed in the furanose form. Spiranic thiohydantoins turned out to be moderate, though selective, inhibitors of glycosidases, whereas their selenium isosters behaved as good free radical scavengers.

Full text of DOI:10.1016/j.tet.2012.03.087

Tetrahedron 68 (2012) 4888e4898
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of conformationally-constrained thio(seleno)hydantoins and
a
-triazolyl
lactones from -arabinose as potential glycosidase inhibitors
D
a
a
,
b
a
ꢀ
ꢀ
*
ꢀ
ꢀ
ꢀ
ꢀ
~
Penelope Merino-Montiel , Oscar Lopez , Eleuterio Alvarez , Jose G. Fernandez-Bolanos
a
ꢀ
Departamento de Química Organica, Facultad de Química, Universidad de Sevilla, Apartado 1203, E-41071 Seville, Spain
Instituto de Investigaciones Químicas “Isla de la Cartuja”, CSIC-USE, Americo Vespucio 49, 41092 Seville, Spain
b
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
We have explored the rich structural diversity provided by an
the source of sugar templates with reduced conformational flexibility. Using transient
a
-azido ester derived from
D
-arabinose as
-thiour-
eido(selenoureido) esters we have prepared spiranic thio(seleno)hydantoins at the C-3 position of the
sugar moiety. In this context, the first example of a stable spiranic -lactam (or aziridinone) was isolated
as a by-product in the hydrogenolysis of the starting -azido ester. Furthermore, using copper(I)-
catalyzed azidoealkyne cycloaddition (click chemistry), we have accessed bicyclic cis-fused -triazolyl
Received 6 February 2012
Received in revised form 16 March 2012
Accepted 20 March 2012
a
a
Available online 28 March 2012
a
a
ꢀ
Dedicated to the memory of Dr. Consolacion
Gasch
lactones fixed in the furanose form. Spiranic thiohydantoins turned out to be moderate, though selective,
inhibitors of glycosidases, whereas their selenium isosters behaved as good free radical scavengers.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Spiranic carbohydrates
Thiohydantoins
Selenohydantoins
Click chemistry reaction
Glycosidase inhibitors
1. Introduction
The number of synthetic carbohydrates containing spiroheter-
ocyclic motifs, not only (thio)hydantoins, has increased incessantly
Incorporation of heterocyclic moieties into carbohydrate-based
scaffolds is a widely used procedure in the preparation of bi-
ologically active compounds, such as immunostimulating agents,1
antitumour2 or antimicrobial derivatives.3
since the isolation of hydantocidin, bearing the heterocyclic motifs
mainly on positions C-1,14 but also on C-2,15 C-316 or C-4;17 position
C-3 is relatively less explored when compared with the anomeric
carbon.
Among the plethora of carbohydrates containing heterocycles
reported so far, a strategy considered for the improvement of the
activities exhibited by such compounds consists in locking the
conformation of the sugar residue as a way of attempting a better
binding with the corresponding targeted biomolecules involved in
their action.4e8 In this context, it is worth mentioning spiro-
annelated and bicyclic-fused carbohydrates. The former emerged
after the isolation in 1991 of hydantocidin, the first known natural
spiro-sugar,9 exhibiting herbicidal and plant regulation activities
with reduced toxicity towards mammals.10 The design of analogous
structures to hydantocidin has allowed the development of very
potent inhibitors of glycogen phosphorylase,11,12 a potential target
in the treatment of type 2 diabetes mellitus.13
2. Results and discussion
In the search for new biologically active carbohydrate de-
rivatives, we have explored the synthesis of conformationally-
constrained architectures hold in locked conformations, and eval-
uated them as potential glycosidase inhibitors.
Herein we have exploited the structural diversity provided by
arabino-configured -azido ester 4, which has been efficiently
transformed into spiro-annelated thiohydantoins, selenohy-
dantoins and an -lactam, together with cis-fused bicyclic -tri-
D-
a
a
a
azolyl lactones. Our main target is to compare the biological activity
of these compounds with the previously reported by us starting
from D
-glucose;18 on the one hand, the compounds presented in
this manuscript have reversed stereochemistry on the spiranic
centre, and on the other hand, the lack of a hydroxymethyl moiety
might confer a different conformational behaviour of the sugar ring
when compared with the glucose counterparts. Therefore these
* Corresponding author. Tel.: þ34 954559997; fax: þ34 954624960; e-mail ad-
ꢀ
ꢀ
dress: osc-lopez@us.es (O. Lopez).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.03.087

P. Merino-Montiel et al. / Tetrahedron 68 (2012) 4888e4898
4889
two aspects covered herein might modify the inhibitory properties
In the literature, reported data on
a
-lactams are scarce;22 such
of the targeted compounds.
compounds are usually obtained by treatment of N-substituted a-
t
Starting from commercially-available
D
-arabinose, the stereo-
halo(mesyloxy)amides with strong bases like BuOK or NaH under
anhydrous conditions;23,24 this family of strained compounds has
received attention because of the regioselective ring-opening re-
actions exerted by certain nucleophiles,23 leading to functionalized
selective synthesis of trichloromethyl derivative 3 has been repor-
ted from the corresponding ulose 2;19 subsequent modified
CoreyeLink reaction20 of 3 with NaN3, and DBU in MeOH furnished
a
-azido ester 4 (Scheme 1), as reported by Sørensen et al.21 Com-
derivatives. Nevertheless, most of a-lactams are highly unstable
pound 4 turned out to be a versatile starting material for the syn-
thesis of an ample number of spiro-annelated and other bicyclic
sugar derivatives, as demonstrated in this contribution.
compounds, and could only be isolated25 when bearing a tertiary
alkyl substituent on the nitrogen atom and at least one tertiary alkyl
group on C-3.26
Surprisingly, aziridinone 6, the first example of a spiranic
a-
lactam, turned out to be a highly stable compound, despite bearing
no substituents on the nitrogen atom, and could even be isolated by
column chromatography and characterized by NMR spectroscopy
without any decomposition. Probably, the prominent steric hin-
drance exerted by the tert-butyldiphenylsilyloxy group, together
with the dioxolane moiety precluded degradation of the
aziridinone.
Attempts to convert aminoester 5 into aziridinone 6 using both
acidic and basic catalysis at different temperatures and solvents
were unsuccessful. Changes in solvent polarity, pH and tempera-
ture of the hydrogenolysis reaction led to no changes in the ratio of
aminoester 5 and aziridinone 6. Based on these results, we propose
that
a-lactam formation takes place via an anionic amide in-
termediate 14 generated in the hydrogenolysis reaction of azide 4.
We postulate that intramolecular acyl nucleophilic substitution
of transient 14 would lead to 6 (Scheme 3, pathway b), whereas
capture of a proton (Scheme 3, pathway a) would lead to the
expected aminoester 5.
Scheme 1.
Catalytic hydrogenation of
yielded a separable mixture of two compounds (Scheme 2):
aminoester 5 (64%) and unexpected spiranic -lactam (or azir-
a-azido ester 4 using Pd/C as catalyst
a
-
a
idinone) 6 (21%). NMR data indicated the lack of the methox-
ycarbonyl group in 6, and a strong deshielding of C-3 (9.7 ppm) in
comparison with
a-aminoester 5; these data are in agreement with
a spirocyclization taking place.16c
Scheme 3.
We attempted the deprotection of aziridinone 6 by treatment
with a TBAF solution; removal of the TBDPS protective group led
not to expected monohydroxylated derivative 15, but to cis-fused
a-
amino- -lactone 16 in an 82% yield. The formation of such a com-
g
pound can be explained considering a spontaneous nucleophilic
attack of C-5 hydroxyl group on the carbonyl group, affording
opening of the lactam moiety (Scheme 4). Lactones are valuable
intermediates in organic synthesis, and in particular, bicyclic car-
bohydrate 1,2-lactones have been subjected to stereoselective
opening with hetero- and C-nucleophiles to furnish functionalized
carbohydrates.27
Scheme 2.

4890
P. Merino-Montiel et al. / Tetrahedron 68 (2012) 4888e4898
showed a deshielding (0.5e2.0 ppm) when compared with C-3 of
aminoester 5, demonstrating the presence of the spiranic ring.
We first attempted partial deprotection of compounds 10 and 11,
which was accomplished using a solution of TBAF, affording the
elimination of the TBDPS group to give monohydroxylated thio-
hydantoins 17 and 18 (Scheme 5) in excellent yields (79 and 78%,
respectively) after chromatographical purification. Fully depro-
tection of N-phenyl thiohydantoin 10 was achieved in two steps:
firstly, treatment with TBAF, and subsequent addition of Amberlite
IR-120(Hþ) resin to the non-isolated monohydroxylated derivative
17 to give 19 with an overall yield of 60%.
Scheme 4.
The structure of amino lactone 16 was further confirmed by X-
ray diffractiony (Fig. 1).
Treatment of aminoester 5 with thiophosgene and CaCO3 in
a mixture of CH2Cl2eH2O afforded isothiocyanato ester 7 (Scheme
2) in a quantitative yield. Aminoester 5 and isothiocyanato ester 7
were coupled with phenyl isothiocyanate and cyclohexylamine,
respectively, to afford spiranic thiohydantoins 10 and 11, re-
spectively (Scheme 2) via the corresponding transient thioureas 8
and 9, which underwent intramolecular acyl nucleophilic sub-
stitution involving the NH group. In the case of thiohydantoin 11,
cyclization took place spontaneously, and thiourea 9 was not
detected by TLC. On the contrary, regarding N-phenyl thio-
hydantoin 10, its formation required addition of Et3N and heating at
60 ꢀC.
Scheme 5.
Moreover, fully deprotection of N-cyclohexyl derivative 11 to
furnish 20 was achieved using two procedures: treatment with
acidic Amberlite IR-120(Hþ) resin (85%) or, alternatively, with
aqueous TFA (68%).
Both deprotected thiohydantoins 19 and 20 were obtained as
anomeric mixtures of pyranosic and furanosic forms (Table 1). The
major isomer of such compounds was assigned to the
a pyranose
form in a 1C4 conformation, based on the J6,7 coupling constant (6.6
and 6.3 Hz, respectively); on the other hand, for the second isomer
in importance, the small value for the same coupling constant
(roughly 3.3 Hz) confirmed the
b anomer. Regarding the two minor
isomers, the one with a higher frequency in the anomeric carbon
(101.1 ppm for 19 and 100.8 ppm for 20) was tentatively assigned28
to the
a furanose form.
Table 1
Anomeric ratio in (CD3)2CO of compounds 19 and 20
Fig. 1. ORTEP drawing of 16.
Compound
a
-Pyranose
b-Pyranose
a
-Furanose
b-Furanose
19
20
15.9
19.1
2.2
3.3
1
1
2.7
NMR data of compounds 10 and 11 showed no signals for the
methoxycarbonyl group; 1H NMR spectra indicated a single signal
for the NH group ranging from 8.27 to 8.89 ppm. Spiranic C-5
da
a
Overlapping of signals.
Theremarkablebiologicalpropertiesexhibited byorganoselenium
compounds, such as antioxidants,29 antimicrobial,30 anticancer31 or
anti-inflammatory agents,32 among others, stimulated us to prepare
easily-available seleno-isosters of the previousspiranicthiohydantoin
templates in order to study their antioxidant properties.
For that purpose, we first attempted the preparation of the
spiranic selenohydantoins by coupling a-isoselenocyanato ester 23
with different amines; such heterocumulene was accessed from
aminoester 5 by formylation (Scheme 6) with freshly-prepared
y
ꢁ
Crystal data for 16: C9H13NO5, M¼215.0, orthorhombic, a¼6.2249(3) A, b¼7.
3
ꢁ
¼90.00ꢀ,
b
¼90.00ꢀ,
g
¼90.00ꢀ, V¼955.36(8) A ,
ꢁ
ꢁ
0817(3) A, c¼21.6720(10) A,
a
T¼173(2) K, space group P2(1)2(1)2(1), Z¼4,
m(Mo K
a
)¼0.123 mmꢁ1, 10,227 re-
flections measured, 1662 independent reflections (Rint¼0.0547). The final R1 values
were 0.0683 (I>2s s(I)). The final R1
(I)). The final wR(F2) values were 0.1911 (I>2
values were 0.0779 (all data). The final wR(F2) values were 0.2010 (all data). The
goodness of fit on F2 was 1.212. CCDC-859845 (16) contains the supplementary
crystallographic data for this paper. These data can be obtained free of charge from
The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/
cif.
acetic-formic anhydride (AFA) in
a
biphasic satd aq

P. Merino-Montiel et al. / Tetrahedron 68 (2012) 4888e4898
4891
NaHCO3eCH2Cl2 medium, following a procedure recently de-
veloped in our research group.33 Formamide 21 was isolated in an
84% yield after chromatographical purification; 1H NMR (8.13 ppm)
and 13C NMR spectra (160.2 ppm) confirmed the presence of the
formamido moiety.
These are the first examples of both, sugar-derived selenohy-
dantoins, and spiranic selenohydantoins.
Attempts to deprotect 26 or 27 failed, and extensive de-
composition was observed, with release of elemental selenium.
We have also exploited the versatility offered by the
a-azido
ester 4 as a building block for the incorporation of triazolyl moieties
via Cu(I)-catalyzed azideealkyne cycloadditions, that is, click
chemistry reactions.34 Triazolyl moiety is a robust scaffold, com-
patible with a plethora of functional groups, and virtually chemi-
cally inert,35 and has found numerous applications in Medicinal
Chemistry, Materials Science, or liquid crystals, among others.
In this context, we have accomplished the preparation of 4-alkyl
and aryl arabino-derived triazoles from 4 using commercially-
available alkynes and sodium ascorbate/CuSO4 as the Cu(I) source
(Scheme 8); triazolyl esters 28e31 were obtained in a 62e85% yield
after chromatographical purification (Table 2).
Scheme 6.
Compound 21 was dehydrated with triphosgene and Et3N, un-
der the same conditions used by our group in the preparation of
sugar-derived isoselenocyanates,33 to give transient non-isolated
isocyanide 22; such a compound was coupled with elemental
black selenium at 90 ꢀC in toluene to furnish
a-isoselenocyanato
ester 23 (Scheme 6) in a modest yield (39%, two steps), coming from
partial decomposition under the reaction conditions.
The presence of the selenium atom was evidenced from the 13C
NMR spectrum, which showed resonance of the eN¼C]Se group
at 134.3 ppm, with a shielding of 6.2 ppm when compared with the
thio-isoster 7, in agreement with isoselenocyanates data.33
Although selenohydantoins can be obtained by direct coupling of
23 with different amines, considering the low yield in the preparation
of the isoselenocyanate, even lower at a higher scale, we decided to
modify the synthetic pathway in order to synthesize selenohydantoins
in a more acceptable overall yield. In that sense, we decided to couple
Scheme 8.
Table 2
Synthesis of
a
-triazolyl esters 28e31
a-aminoester 5 with p-tolyl and
a
-naphthyl isoselenocyanates.33
Entry
Alkyne Triazole
Compd
Yielda (%)
Such aryl isoselenocyanates were coupled with 5 under mild
conditions to furnish transient non-detected selenoureas 24 and 25,
which underwent a spirocyclization involving the methoxycarbonyl
group leading to spiroselenohydantoins 26 and 27 (Scheme 7). The
absence of signals in 1H and 13C NMR spectra regarding the
methoxycarbonyl group confirms the cyclization reaction.
1
2
28
62
29
66
3
30
31
85
76
4
a
Isolated yield.
Scheme 7.

4892
P. Merino-Montiel et al. / Tetrahedron 68 (2012) 4888e4898
Carbohydrates bearing a triazolyl moiety on C-3 of
D
-xylo,
D
-
-
galacto, -allo and -gluco configurations were reported to be
D
D
a
glucosidase inhibitors, even bearing protective groups on the hy-
droxyl groups.36 In our case, it was our intention to study how
a bicyclic lactone could modulate the biological activity.
D
1H NMR spectra of compounds 28e31 showed a singlet in the
range 8.02e7.52 ppm, which is consistent with the proton H-5 of
the triazolyl moiety, in agreement with reported data on triazole-
containing carbohydrates.36
C
Triazole 28 was treated with a solution of TBAF in THF in order to
obtain partially-deprotected derivative 36 (Scheme 9). Neverthe-
less, the 1H NMR spectrum showed absence of the methox-
ycarbonyl moiety, and a remarkable difference between coupling
constants J4,5a and J4,5b; these data suggest instead the formation of
cisecis-arranged tricyclic lactone 37, resulting from nucleophilic
attack of C-5 hydroxyl group on the carbonyl group, maybe cata-
lyzed by the TBAF present in the reaction medium.
B
A
Fig. 2. Selected 1H NMR regions for the deprotection of 28.
Scheme 9.
The only two bibliographic reports concerning the synthesis of
lactones on C-3 of a furanose involve either an intramolecular nu-
cleophilic addition of an alkoxide on a cyano group, followed by
hydrolysis of the transient imine,37 or the intramolecular conden-
sation between hydroxyl and amido groups.38
Unfortunately, attempts to purify derivative 37 from TBAF by
column chromatography were unsuccessful.
Scheme 10.
Alternatively, we tried to eliminate tetrabutylammonium salts
following a recent reported procedure, based on treatment with
CaCO3 and Dowex 50W resin;39 nevertheless, although the ratio of
the tetrabutylammonium was reduced, it could not be eliminated
completely. Subsequently, in order to enable the purification pro-
cess, we decided to remove simultaneously both, the TPDPS and
isopropylidene groups, using an aqueous TFA solution.
Treatment of triazole 28 with a 9:1:1 TFAeH2OeTHF mixture at
rt afforded a complex mixture, as indicated by NMR spectrum
(Fig. 2A); the presence of a series of singlets in the region
3.60e3.75 ppm, presumably corresponding to methoxycarbonyl
groups suggests the existence of anomeric mixtures of non-
lactonized pyranosic and furanosic forms 38 and 39, together
with bicyclic lactone 32 (Scheme 10).
This successful procedure, yielding exclusively cis-fused bicyclic
lactones, was also applied to triazoles 29e31, under the same re-
action conditions, to afford unprotected a-triazolyl lactones 33e35,
from moderate to excellent yields (Table 3), as anomeric mixtures
(Scheme 8) in roughly 1:1 ratio (Table 4).
Coupling constant J1,2 for the
0 Hz in all the unprotected triazolyl lactones, whereas for the
isomer it ranged from 3.3 to 4.2 Hz. The unequivocal assignment
a anomer was found to be roughly
b
of the complete spin system for both anomers was accomplished
using HMBC experiments. The strong differences found for cou-
pling constants J4,5a, J4,5b in 32e35 (J4,5aw6 Hz, J4,5b 0e2.7 Hz) when
In the deprotection of 28, a gradual increase of temperature led
to a simplification of the 1H NMR spectrum in the sugar region
(5.30e4.60 ppm), and to the disappearance of the signals around
3.60e3.75 ppm of the methoxycarbonyl group (Fig. 2BeD). In an
additional assay, triazole 28 was heated at 50 ꢀC in acidic medium
for 20 h; subsequent purification by gel permeation chromatogra-
phy (Biogel P2) afforded exclusively unprotected triazolyl lactone
32 as a 1:1 anomeric mixture (Scheme 8).
compared
with
non-lactonized
derivatives
28e31
(J4,5a¼J4,5b¼6.3e6.2 Hz) are compatible with the lack of confor-
mational freedom characteristic of a bicyclic system.
Regarding 13C NMR spectra, carbonyl groups of lactones 32e35
(181.2e169.8 ppm) showed deshielding when compared with their
ester counterparts 28e31 (166.0e165.1 ppm); similarly, C-5 of the
unprotected triazolyl lactones also showed a deshielding of roughly
10 ppm compared with protected compounds.

P. Merino-Montiel et al. / Tetrahedron 68 (2012) 4888e4898
4893
Table 3
a stable commercially-available free radical, which constitutes the
basis for one of the most popular assays for testing the radical-
scavenging activity.40 The methanolic solution of this compound,
of a deep purple colour, exhibits a strong absorption at 515 nm,
which is reduced in the presence of an antioxidant agent to give
a yellowish solution. The remaining concentration of DPPH is,
therefore, inversely proportional to the free radical-scavenging
activity exhibited by the antioxidant agent.41
Deprotection of compounds 28e31
Compound
Triazole
Yielda (%)
28
86
The calculated EC50 value for spiranic selenohydantoin 27, that
is, the concentration of the antioxidant agent required for reducing
50% of the initial DPPH concentration, was found to be 87ꢂ15
mM;
this value is quite similar to that reported40 for BHT (butylhy-
droxytoluene or 2,6-di-tert-butyl-4-methylphenol), a synthetic in-
29
50
dustrial antioxidant agent (EC50¼60
mM).
4. Conclusions
In conclusion, we have demonstrated the high chemical versa-
tility of a -arabino-configured -azido ester template by efficiently
D
a
transforming it into novel conformationally-locked structures with
potential biological activity: spiranic thio- and selenohydantoins,
30
31
64
36
via spirocyclization involving an
or bicyclic cis-fused -triazolyl lactones, through click chemistry
reactions. An hitherto unknown and stable spiranic -lactam was
also isolated as a by-product. One of the thiohydantoins behaved as
a moderate inhibitor of -galactosidase, whereas protected sele-
a-thioureido or selenoureido ester,
a
a
a
nohydantoins behaved as good free radical scavengers.
5. Experimental section
a
Isolated yield.
5.1. General procedures
Optical rotations were measured with a Jasco P-2000 polarim-
eter. 1H (300 and 500 MHz) and 13C (75.5 and 125.7 MHz) NMR
spectra were recorded on Bruker Avance-300 and Avance-500
spectrometers and the indicated spectra data were registered at
rt. The assignments of 1H and 13C signals were confirmed by ho-
monuclear COSY, and heteronuclear 2D correlated spectra, re-
spectively. Mass spectra (CI and LSI) were recorded on Micromass
AutoSpec-Q mass spectrometers with a resolution of 1000 or
10,000 (10% valley definition). For LSI spectra, ions were produced
by a beam of xenon atoms and Csþ ions, respectively, using thio-
glycerol as matrix and NaI as additive. TLC was performed on alu-
minium pre-coated sheets (E. Merck Silica Gel 60 F254); spots were
visualized by UV light, by charring with 10% H2SO4 in EtOH, with 3%
ninhydrin in EtOH or with vanillin (1.5% in EtOH in the presence of
1% H2SO4). Column chromatography was performed using E. Merck
3. Biological activities
3.1. Glycosidase inhibition
Thiohydantoins 19, 20 and
a
-triazolyl lactones 32e35 were
tested as potential glycosidase inhibitors against five commercially-
available glycosidases ( -glucosidase from baker’s yeast, -gluco-
sidase from almonds, -galactosidase from green coffee beans,
galactosidase from Aspergillus oryzae and -galactosidase from
a
a
b
b-
b
Escherichia coli), and against glycogen phosphorylase type b from
rabbit muscle.
Cyclohexyl thiohydantoin 20 was found to be a moderate,
though selective, inhibitor of
phenyl derivative 19 exhibited weak inhibition against glycogen
phosphorylase (Ki¼910 M). Regarding triazole derivatives, only
cyclohexenyl derivative 33 exhibited very weak inhibition against
-glucosidase (10% inhibition at 404 M). These results, although
a
-galactosidase (Ki¼160
mM), and
Silica Gel 60 (40e63 mm).
m
5.1.1. 3-Amino-3-deoxy-5-O-tert-butyldiphenylsilyl-1,2-O-iso-
propylidene-3-C-methoxycarbonyl- -arabinofuranose (5) and
a
m
b-D
moderate, are much better than those obtained for glucose
counterparts.18
(3R,4S,6S,7S)-4-tert-butyldiphenylsilyloxymethyl-6,7-(dimethylme-
thylenedioxy)-5-oxa-1-azaspiro[2.4]heptan-2-one (6). To a solution
of azidoester 4 (300 mg, 0.59 mmol) in methanol (23 mL) was
added 10% Pd/C (90 mg) and the mixture was hydrogenated at at-
mospheric pressure and rt for 30 min. Then, the reaction was fil-
tered over a Celite pad and the filtrate was concentrated to dryness
to give a mixture of compounds 5 and 6 that were separated by
column chromatography (9:1/3:2 hexaneeEtOAc).
3.2. Antioxidant activity
We tested the radical-scavenging activity of spiranic selenohy-
dantoin 27. In this context, DPPH (1,1-diphenyl-2-picrylhydrazyl) is
Eluted first was 6: 56 mg, 21%; Rf 0.74 (3:2 hexaneeEtOAc); ½a D20
ꢃ
Table 4
Anomeric ratio in compounds 32e35
þ10 (c 1.16, CH2Cl2); IR nmax 3461, 3009, 2970, 1739, 1439, 1365,
1228, 1208 cmꢁ1 1H NMR (300 MHz, CDCl3):
; d 10.58 (s, 1H, NH),
Compd
32
33
1:1a
34
35
a
/
b
ratio
1:1a
1:1a
1:1a
7.63e7.57 (m, 4H, AreH), 7.42e7.35 (m, 6H, AreH), 6.06 (d, 1H,
3:1b
0
J6,7¼4.2 Hz, H-6), 4.82 (d, 1H, H-7), 4.56 (dd, 1H, J4,1 a¼9.4 Hz,
J4,1 b¼4.3 Hz, H-4), 4.20 (t, 1H, J1 a,1 b¼9.3 Hz, H-10a), 3.84 (dd, 1H, H-
a
0
0
0
In (CD3)2CO.
In DMSO-d6.
10b), 1.59, 1.36 (2s, 3H each, 2CH3), 1.00 (s, 9H, C(CH3)3); 13C NMR
b

4894
P. Merino-Montiel et al. / Tetrahedron 68 (2012) 4888e4898
(75.5 MHz, CDCl3):
d
172.0 (C-2), 135.6, 132.8, 132.7, 130.1, 130.0,
1.5 h. After that, the solvent was removed under reduced pressure
and the residue was purified by column chromatography (9:1
hexaneeEtOAc) to give thiohydantoin 11 as a foam: 84 mg, quant.;
128.0, 127.0 (AreC), 116.5 (CMe2), 106.2 (C-6), 85.5 (C-7), 81.5 (C-4),
77.4 (C-3), 61.3 (C-10), 27.4, 27.1 (2CH3), 26.7 (C(CH3)3), 19.2
(C(CH3)3); Anal. Calcd for C25H31NO5Si: C, 66.20; H, 6.89; N, 3.09,
found: C, 66.27; H, 6.95; N, 3.30.
Rf 0.31 (4:1 hexaneeEtOAc); ½a D23
ꢃ
ꢁ20 (c 0.99, CH2Cl2); 1H NMR
(500 MHz, CDCl3):
d 8.27 (s, 1H, NH), 7.67e7.62 (m, 4H, AreH),
Eluted second was 5: 182 mg, 64%; Rf 0.34 (3:2 hexaneeEtOAc);
7.41e7.37 (m, 6H, AreH), 5.86 (d, 1H, J8,9¼4.0 Hz, H-8), 4.65 (d, 1H,
½
a 2D4
ꢃ
ꢁ13 (c 1.02, CH2Cl2); 1H NMR (300 MHz, CDCl3):
d 7.65e7.60 (m,
H-9), 4.45 (tt, 1H, JH,H¼4.0 Hz, JH,H¼12.5 Hz, H-100), 4.28 (dd, 1H,
J6,1 a¼7.5 Hz, J1 a,1 b¼10.5 Hz, H-10a), 4.19 (dd, 1H, J6,1 b¼5.0 Hz, H-6),
3.84 (dd, 1H, H-10b), 2.30 (dq, 1H, JH,H¼12.8 Hz, JH,H¼4.0 Hz, CHcy-
clohex.), 2.22 (dq,1H, JH,H¼13.0 Hz, JH,H¼4.0 Hz, CHcyclohex.),1.85e1.59
(m, 6H, CHcyclohex.), 1.46, 1.28 (2s, 3H each, 2CH3), 1.24e1.18 (m, 2H,
0
0
0
0
4H, AreH), 7.42e7.34 (m, 6H, AreH), 5.84 (d, 1H, J1,2¼4.0 Hz, H-1),
4.49 (d, 1H, H-2), 4.16 (dd, 1H, J4,5a¼7.8 Hz, J5a,5b¼9.1 Hz, H-5a), 4.07
(dd,1H, J4,5b¼5.7 Hz, H-4) 3.96 (dd,1H, H-5b), 3.70 (s, 3H, OMe), 3.40
(br s, 2H, NH2), 1.43, 1.30 (2s, 3H each, 2CH3), 1.03 (s, 9H, (C(CH3)3));
13C NMR (75.5 MHz, CDCl3):
d
171.4 (CO),135.6 (ꢄ4),133.0 (ꢄ2),129.9
2CHcyclohex.), 1.03 (C(CH3)3); 13C NMR (125.7 MHz, CDCl3):
d 183.4
(ꢄ2),127.9 (ꢄ4) (AreC),115.6 (CMe2),104.9 (C-1), 90.1 (C-2), 85.7 (C-
4), 67.7 (C-3), 63.2 (C-5), 52.1 (OMe), 27.1 (CH3), 26.9 (ꢄ4) (CH3,
C(CH3)3), 19.2 (C(CH3)3); CIMS m/z 486 ([MþH]þ, 13%); HRCI-MS
calcd for C26H36NO6Si ([MþH]þ): 486.2312, found: 486.2298.
(C-2), 167.6 (C-4), 135.7 (ꢄ2), 133.2, 133.1, 129.9 (ꢄ2), 127.9 (ꢄ2)
(AreC), 115.7 (CMe2), 105.1 (C-8), 87.2 (C-9), 85.5 (C-6), 68.2 (C-5),
62.3 (C-10), 55.9 (C-100), 28.9, 28.7 (2CH2), 27.0 (C(CH3)), 26.8, 26.5
(2CH3), 26.0 (ꢄ2), 25.2 (2CH2), 19.3 (C(CH3)3); LSIMS m/z 617
([MþNa]þ, 44%); HRLSI calcd for C32H42N2NaO5SSi ([MþNa]þ):
617.2481, found: 617.2488.
5.1.2. 5-O-tert-Butyldiphenylsilyl-3-deoxy-3-isothiocyanato-1,2-O-iso-
propylidene-3-C-methoxycarbonyl-b-D-arabinofuranose (7). To a solu-
tion of aminoester 5 (37 mg, 0.08 mmol) in a 1:1 mixture of
CH2Cl2eH2O (4 mL), were added CaCO3 (31.5 mg, 0.31 mmol) and
thiophosgene (7.8 mL, 0.10 mmol), and the mixture was vigorously
5.1.5. 3-Amino-3-C-3,5-carbolactono-3-deoxy-1,2-O-isopropylidene-
b-D
-arabinofuranose (16). To a solution of aziridinone 6 (230 mg,
0.51 mmol) in THF (2 mL) was added a 1 M solution of TBAF in THF
(560 L, 0.56 mmol), and the mixture was stirred at rt for 2 days.
stirred for 20 min. Then, the salts were filtered off and the phases
were separated. The aqueous phase was extracted with CH2Cl2
(2ꢄ25 mL), and the combined organic fractions were dried over
MgSO4, filtered and the filtrate was concentrated to dryness.
The residue was purified by column chromatography (19:1
hexaneeEtOAc) to give isothiocyanate 7 as a syrup: 40 mg, 99%; Rf
m
Then, the solvent was removed under reduced pressure, and the
residue was purified by column chromatography (4:1 hex-
aneeEtOAc/EtOAc) to give lactone 16 as an amorphous solid,
which was recrystallized from ethanol: 91 mg, 82%; mp:
196e197 ꢀC; Rf 0.24 (7:3 hexaneeEtOAc); ½a 2D6
ꢁ99 (c 0.56,
ꢃ
0.58 (4:1 hexaneeEtOAc); ½a D24
ꢃ
ꢁ36 (c 1.25, CH2Cl2); 1H NMR
CH2Cl2); IR nmax 3440, 3014, 2969, 1738, 1370, 1222, 1139, 1105,
(300 MHz, CDCl3):
d
7.68e7.64 (m, 4H, AreH), 7.44e7.36 (m, 6H,
943 cmꢁ1; 1H NMR (500 MHz, DMSO-d6):
H-1), 4.64 (dd, 1H, J4,5a¼7.9 Hz, J4,5b¼5.7 Hz, H-4), 4.46 (d, 1H, H-
2), 4.42 (dd, 1H, J5a,5b¼9.7 Hz, H-5a), 4.06 (dd, 1H, H-5b), 2.47 (s,
2H, NH2), 1.44, 1.24 (2s, 3H each, 2CH3); 13C NMR (125.7 MHz,
d
6.06 (d, 1H, J1,2¼3.5 Hz,
AreH), 5.85 (d, 1H, J1,2¼3.6 Hz, H-1), 4.79 (d, 1H, H-2), 4.24 (dd, 1H,
J4,5a¼7.2 Hz, J4,5b¼6.0 Hz, H-4), 4.15 (dd,1H, J5a,5b¼10.2 Hz, H-5a) 3.95
(dd, 1H, H-5b), 3.78 (s, 3H, OMe), 1.43, 1.33 (2s, 3H each, 2CH3), 1.06
(s, 9H, C(CH3)3); 13C NMR (75.5 MHz, CDCl3):
d
164.5 (C]O), 140.5
DMSO-d6): d 175.2 (CO), 112.5 (CMe2), 108.4 (C-1), 87.3 (C-2), 86.3
(NCS),135.7 (ꢄ2), 133.2,133.0,129.9, 127.9 (ꢄ2) (AreC), 116.4 (CMe2),
104.8 (C-1), 88.6 (C-2), 86.5 (C-4), 72.5 (C-3), 62.6 (C-5), 53.1 (OMe),
27.2 (CH3), 26.8 (C(CH3)3), 26.8 (CH3), 19.3 (C(CH3)3); LSIMS m/z 550
([MþNa]þ, 78%); HRLSI-MS calcd for C27H33NNaO6SSi ([MþNa]þ):
550.1696, found: 550.1701.
(C-4), 69.7 (C-5), 68.7 (C-3), 26.4, 25.6 (2CH3); CIMS m/z 216
([MþH]þ, 12%); HRCI-MS calcd for C9H14NO5 ([MþH]þ): 216.0872,
found: 216.0872; Anal. Calcd for C9H13NO5: C, 50.23; H, 6.09; N,
6.51, found: C, 50.22; H, 6.08; N, 6.19.
5.2. General procedure for the partial deprotection of
spirothiohydantoins 10 and 11
5.1.3. (5R,6S,8S,9S)-6-tert-Butyldiphenylsilyloxymethyl-8,9-(dime-
thylmethylenedioxy)-3-phenyl-2-thioxo-7-oxa-1,3-diazaspiro[4.4]
nonan-4-one (10). To
0.23 mmol) in THF (1.5 mL) was added phenyl isothiocyanate (41
0.34 mmol) and the mixture was stirred at rt for 16 h. After disap-
a
solution of aminoester
5
(112 mg,
To a solution of thiohydantoin 10 or 11 (0.16 mmol) in THF
(2.0 mL) was added a 1 M solution of TBAF in THF (190 mL,
0.19 mmol) and the mixture was stirred at rt during the time in-
dicated in each case. Then, the solvent was removed under reduced
pressure and the residue was purified by column chromatography
(4:1/3:2 hexaneeEtOAc) to give compounds 17 and 18 as foams.
mL,
pearance of the starting material, triethylamine (16 mL, 0.11 mmol)
was added, and the solution was heated at 60 ꢀC for 1 h. Then, the
solvent was removed under reduced pressure and the residue was
purified by column chromatography (4:1 hexaneeEtOAc) to give
thiohydantoin 10 as a foam: 123 mg, 91%; Rf 0.65 (3:2 hex-
5. 2.1. (5R, 6S, 8S, 9S)-8, 9-(Dimethylmethylenedioxy)-6-
hydroxymethyl-3-phenyl-2-thioxo-7-oxa-1,3-diazaspiro[4.4]nonan-
4-one (17). Stirring was kept for 24 h: 46 mg, 81%; Rf 0.10 (7:3
aneeEtOAc); ½a 2D3
ꢃ
ꢁ36 (c 0.57, CH2Cl2); 1H NMR (300 MHz, CDCl3):
d
8.89 (s, 1H, NH), 7.66e7.61 (m, 4H, AreH), 7.45e7.28 (m, 11H,
AreH), 5.88 (d,1H, J8,9¼3.6 Hz, H-8), 4.84 (d,1H, H-9), 4.33 (m, 2H, H-
hexaneeEtOAc); ½a D23
ꢃ
ꢁ76 (c 1.30, CH2Cl2); 1H NMR (300 MHz,
6, H-10a), 3.87 (dd, 1H, J6,1 b¼3.3 Hz, J1 a,1 b¼9.0 Hz, H-10b), 1.47, 1.31
CD3OD):
d 7.53e7.41 (m, 3H, AreH), 7.32e7.28 (m, 2H, AreH), 5.96
0
0
0
(2s, 3H each, 2CH3),1.00 (s, 9H, C(CH3)3); 13CNMR (75.5 MHz, CDCl3):
(d, 1H, J8,9¼3.9 Hz, H-8), 4.85 (d, 1H, H-9), 4.21 (t, 1H,
J6,1 a¼J6,1 b¼7.0 Hz, H-6), 3.93 (m, 2H, H-10a, H-10b), 1.58, 1.25 (2s, 3H
0
0
d
182.6 (C-2),166.9 (C-4),135.7, 133.0,132.9,132.6,130.0 (ꢄ2),129.4,
129.1,128.6,128.0,127.9 (AreC),116.1 (CMe2),105.1 (C-8), 87.7 (C-9),
84.9 (C-6), 69.9 (C-5), 62.0 (C-10), 27.0, 26.7 (2CH3, C(CH3)3), 19.3
(C(CH3)); LSIMS m/z 611 ([MþNa]þ, 17%); HRLSI-MS calcd for
C32H36N2NaO5SSi ([MþNa]þ): 611.2012, found: 611.2026.
each, 2CH3); 13C NMR (75.5 MHz, CD3OD):
d 183.8 (C-2), 170.0 (C-4),
134.9, 130.2, 130.1, 129.9 (AreC), 119.9 (CMe2), 106.6 (C-8), 88.6 (C-
9), 85.6 (C-6), 77.1 (C-5), 61.3 (C-10), 28.0 (ꢄ2) (2CH3); LSIMS m/z
373 ([MþNa]þ, 48%); HRLSI-MS calcd for C16H18N2NaO5S
([MþNa]þ): 373.0834, found: 373.0832.
5.1.4. (5R,6S,8S,9S)-6-tert-Butyldiphenylsilyloxymethyl-3-
cyclohexyl-8,9-(dimethylmethylenedioxy)-2-thioxo-7-oxa-1,3-
diazaspiro[4.4]nonan-4-one (11). To a solution of isothiocyanato
ester 7 (75 mg, 0.14 mmol) in THF (1.5 mL) was added cyclohex-
5.2.2. (5R,6S,8S,9S)-3-Cyclohexyl-8,9-(dimethylmethylenedioxy)-6-
hydroxymethyl-2-thioxo-7-oxa-1,3-diazaspiro[4.4]nonan-4-one
(18). Stirring was kept for 48 h: 46 mg, 81%; Rf 0.11 (7:3 hex-
ylamine (24
mL, 0.21 mmol) and the mixture was stirred at rt for
aneeEtOAc); ½a 2D3
ꢃ
ꢁ70 (c 1.25, CH2Cl2); 1H NMR (300 MHz, CDCl3):

P. Merino-Montiel et al. / Tetrahedron 68 (2012) 4888e4898
4895
d
5.97 (d, 1H, J8,9¼3.6 Hz, H-8), 4.67 (d, 1H, H-9), 4.46 (tt, 1H,
a
furan:
d
5.50 (d, 1H, J8,9¼5.1 Hz, H-8), 4.29 (d, 1H, H-9);
b
furan:
JH,H¼3.7 Hz, JH,H¼12.3 Hz, H-100), 4.27 (t,1H, J6,1 a¼J6,1 b¼6.5 Hz, H-6),
d
5.34 (d, 1H, J8,OH¼6.6 Hz, OH-8), 5.16 (m, 1H, H-8), 3.91 (m, 1H, H-
pyran:
184.7 (C-2), 174.5 (C-4), 95.0 (C-7), 72.1 (C-6), 69.8 (C-5), 68.8 (C-
10), 64.8 (C-9), 55.4 (C-10), 29.2, 29.1, 26.7, 26.6, 25.9 (5CH2);
py-
ran: 184.3 (C-2), 176.9 (C-4), 94.4 (C-7), 70.8 (C-5), 68.4 (C-10),
68.2 (C-6), 61.3 (C-9), 55.7 (C-10), 29.1, 26.5, 25.9 (3CH2);
furan:
0
0
4.12 (m, 1H, H-10a), 3.91 (dd, 1H, J1 a,1 b¼11.7 Hz, H-10b), 2.34e2.03
(m, 2H, CH2), 1.86e1.82 (m, 2H, CH2), 1.68e1.65 (m, 3H, CH2, CHcy-
clohex.), 1.65, 1.32 (2s, 3H each, 2CH3), 1.40e1.21 (m, 3H, CH2, CHcy-
10a), 3.56 (m, 1H, H-10b); 13C NMR (75.5 MHz, (CD3)2CO):
a
0
0
d
b
clohex.); 13C NMR (75.5 MHz, CDCl3):
d
183.2 (C-2), 168.9 (C-4), 115.3
d
(CMe2),105.6 (C-8), 86.4 (C-9), 86.0 (C-6), 68.4 (C-5), 61.5 (C-10), 56.1
(C-100), 28.6 (CH2), 28.5 (CH3), 26.4 (CH2), 26.3 (CH2), 26.0 (ꢄ2) (CH2,
CH3), 25.2 (CH2); LSIMS m/z 379 ([MþNa]þ, 100%); HRLSI-MS calcd
for C16H24N2NaO5S ([MþNa]þ): 379.1304, found: 379.1309.
a
d
100.8 (C-8); b furan: d
92.6 (C-8), 59.3 (C-10); LSIMS m/z 339
([MþNa]þ, 29%); HRLSI-MS calcd for C13H20N2NaO5S ([MþNa]þ):
339.0991, found: 339.0982.
5.2.3. (5R,6S,7R and 7S,10S)-6,7,10-Trihydroxy-3-phenyl-2-thioxo-8-
oxa-1,3-diazaspiro[4.5]decan-4-one (19p) and (5R,6S,8R and 8S,9S)-
8,9-dihydroxy-6-hydroxymethyl-3-phenyl-2-thioxo-7-oxa-1,3-
diazaspiro[4.4]nonan-4-one (19f). To a solution of thiohydantoin 10
(121 mg, 0.20 mmol) in THF (2 mL) was added a 1 M solution of
5.2.5. 5-O-tert-Butyldiphenylsilyl-3-deoxy-3-formamido-1,2-O-iso-
propylidene-3-C-methoxycarbonyl-
lution of aminoester 5 (102 mg, 0.21 mmol) in a 1:1 mixture of satd
aq NaHCO3eCH2Cl2 (5 mL) was added freshly-prepared AFA (86 L,
b-D-arabinofuranose (21). To a so-
m
0.64 mmol), and the mixture was vigorously stirred for 2.5 h. Then,
the phases were separated and the aqueous phase was extracted
with CH2Cl2 (2ꢄ10 mL); the combined organic fractions were dried
over MgSO4, filtered, and the filtrate was concentrated to dryness.
The residue was purified by column chromatography (4:1/2:1
hexaneeEtOAc) to give formamide 21 as a foam: 91 mg, 84%; Rf 0.29
TBAF in THF (250 mL, 0.25 mmol) and the mixture was stirred at rt
for 24 h. Then, H2O (2 mL) and Amberlite IR-120(Hþ) resin
(164 mg) were added and the mixture was stirred at rt for 36 h.
After that, the resin was filtered off, and the filtrate was concen-
trated to dryness; the residue was purified by gel permeation
a 23
chromatography (Biogel P2) to give thiohydantoin 19 as an
a/
(3:2 hexaneeEtOAc); ½ ꢃD ꢁ24 (c 0.83, CH2Cl2); 1H NMR (300 MHz,
b
mixture of pyranose and furanose forms: 38 mg, 60%; Rf 0.45
CDCl3):
d
8.13 (d, 1H, JNH,CHO¼1.2 Hz, CHO), 7.61e7.56 (m, 4H, AreH),
(EtOAc); ½a 2D5
ꢃ
ꢁ38 (c 1.09, MeOH); 1H NMR (300 MHz, CD3OD):
7.46e7.40 (m, 6H, AreH), 6.82 (s,1H, NH), 6.05 (d,1H, J1,2¼3.9 Hz, H-
1), 5.16 (d, 1H, H-2), 4.71 (dd, 1H, J4,5a¼6.6 Hz, J4,5b¼8.1 Hz, H-4),
4.00 (m, 2H, H-5a, H-5b), 3.76 (s, 3H, OMe), 1.50, 1.36 (2s, 3H each,
d
d
7.51e7.38 (m, 3H, AreH), 7.33e7.25 (m, 2H, AreH); a pyranose:
5.19 (d, 1H, J6,7¼6.6 Hz, H-7), 4.18 (dd, 1H, J9a,10¼1.8 Hz,
J9a,9b¼12.3 Hz, H-9a), 4.12 (dd, 1H, J9b,10¼3.3 Hz, H-10), 3.99 (d, 1H,
H-6), 3.89 (dd, 1H, H-9b); pyranose:
5.13 (d, 1H, J6,7¼3.3 Hz, H-
7), 4.29 (m, 1H, H-6), 4.23e4.20 (m, 1H, J9a,10¼2.4 Hz, H-9a), 3.72
(dd, 1H, J9b,10¼4.2 Hz, J9a,9b¼12.9 Hz, H-9b); furanose: 5.55 (d,
furanose: 5.24 (d, 1H,
2CH3), 1.02 (s, 9H, C(CH3)3); 13C NMR (75.5 MHz, CDCl3):
d 168.5
b
d
(CO), 160.2 (CHO) 135.6 (ꢄ2), 133.0, 132.8, 130.0, 127.3 (AreC), 116.7
(CMe2), 104.9 (C-1), 88.0 (C-2), 80.5 (C-4), 68.5 (C-3), 63.1 (C-5), 52.6
(OMe), 27.7, 27.2 (2CH3), 26.8 (C(CH3)3), 19.2 (C(CH3)3); CIMS m/z
414 ([MþH]þ, 2%); HRCI-MS calcd for C27H36NO7Si ([MþH]þ):
514.2261, found: 514.2237.
a
d
1H, J8,9¼5.4 Hz, H-8), 4.44 (d, 1H, H-9),
b
d
J8,9¼3.3 Hz, H-8), 4.09e4.04 (m, 1H, H-10a), 3.93 (d, 1H, H-9),
3.66e3.62 (m, 1H, H-10b); 13C NMR (75.5 MHz, CD3OD):
a
pyra-
nose:
95.3 (C-7), 72.3 (C-6), 71.8 (C-5), 69.0 (C-10), 65.1 (C-9);
nose: 94.4 (C-7), 68.4 (C-10), 68.2 (C-6), 61.7 (C-9);
101.1;
d
184.2 (C-2), 173.7 (C-4), 134.8, 130.4, 129.5, 129.4 (AreC),
5.2.6. 5-O-tert-Butyldiphenylsilyl-3-deoxy-3-isoselenocyanato-1,2-
b
pyra-
O-isopropylidene-3-C-methoxycarbonyl-
b-D-arabinofuranose
d
a furanose:
(23). To a mixture of formamide 21 (28 mg, 0.05 mmol), Et3N
92.6 (C-8), 69.8 (C-9), 59.3 (C-10); LSIMS m/z
(38.5 mL, 0.27 mmol) and 4 A molecular sieves in anhydrous tol-
ꢁ
d
b
furanose:
d
311 ([MþNa]þ, 16%); HRLSI-MS calcd for C13H15N2O5S ([MþH]þ):
uene (1 mL) was dropwise added a solution of triphosgene (13 mg,
0.04 mmol) at 0 ꢀC and inert atmosphere during a period of
30 min. After that, the mixture was kept stirring at 0 ꢀC for further
15 min, and heated at 90 ꢀC for 3.5 h; a less polar compound was
observed by TLC, corresponding to transient isocyanide. Then,
311.0702, found: 311.0690.
5.2.4. (5R,6S,7R and 7S,10S)-3-Cyclohexyl-6,7,10-trihydroxy-2-
thioxo-8-oxa-1,3-diazaspiro[4.5]decan-4-one (20p) and (5R,6S,8R
and 8S,9S)-3-cyclohexyl-8,9-dihydroxy-6-hydroxymethyl-2-thioxo-
7-oxa-1,3-diazaspiro[4.4]nonan-4-one (20f). Method A: To a solu-
tion of thiohydantoin 18 (47 mg, 0.13 mmol) in a 1:1 THFeH2O
mixture (2 mL) was added Amberlite IR-120(Hþ) resin (104 mg).
The mixture was stirred at 50 ꢀC for 24 h. Then, the resin was fil-
tered off and the filtrate was concentrated to dryness to give thi-
black selenium (27 mg, 0.35 mmol) and Et3N (7.6 mL, 0.05 mmol)
were added, and the corresponding mixture was heated at 90 ꢀC in
the darkness for 36 h. After that, the reaction was diluted with
CH2Cl2 (5 mL), the salts were filtered off on a Celite pad, and the
filtrate was concentrated to dryness. The residue was purified by
column chromatography (19:1 hexaneeEtOAc) to give iso-
selenocyanate 23 as a foam: 12 mg, 39%; Rf 0.65 (4:1 hexane-
ohydantoin 20 as an amorphous solid, and as an a/b mixture of
pyranose and furanose forms: 34 mg (85%).
EtOAc); ½a 2D4
ꢃ
ꢁ92 (c 0.46, CH2Cl2); 1H NMR (300 MHz, CDCl3):
Method B: To a solution of thiohydantoin 18 (45 mg, 0.12 mmol)
in a 1:1 THFeH2O mixture (1 mL) was added TFA (4.5 mL), and the
mixture was stirred at rt for 6 h. Then, the solvent was removed
under reduced pressure and co-evaporated with toluene several
times. The residue was purified by gel permeation chromatography
(Biogel P2) to give thiohydantoin 20 as an amorphous solid, and as
d 7.67e7.63 (m, 4H, AreH), 7.44e7.36 (m, 6H, AreH), 5.86 (d, 1H,
J1,2¼3.6 Hz, H-1), 4.86 (d, 1H, H-2), 4.27 (dd, 1H, J4,5a¼7.8 Hz,
J4,5b¼6.0 Hz, H-4), 4.16 (dd, 1H, J5a,5b¼10.2 Hz, H-5a) 3.94 (dd, 1H,
H-5b), 3.79 (s, 3H, OMe), 1.43, 1.34 (2s, 3H each, 2CH3), 1.05 (s, 9H,
C(CH3)3); 13C NMR (75.5 MHz, CDCl3):
d 163.5 (CO), 135.8, 135.7
(AreC), 134.3 (NCSe), 133.1, 132.9, 130.0 (ꢄ2), 128.0, 127.9 (AreC),
116.9 (CMe2), 104.8 (C-1), 88.9 (C-2), 85.9 (C-4), 72.4 (C-3), 62.3 (C-
5), 53.3 (OMe), 27.4 (ꢄ2) (2CH3), 26.9 (C(CH3)3), 19.3 (C(CH3)3);
an a/b mixture of pyranose and furanose forms: 26 mg, 68%; Rf 0.50
(EtOAc); ½a 2D4
ꢃ
ꢁ42 (c 1.16, (CH3)2CO); 1H NMR (300 MHz, (CD3)2CO)
a
pyran:
d
8.87 (br s, 1H, NH), 5.14 (d, 1H, J6,7¼6.3 Hz, H-7), 4.66 (m,
LSIMS m/z 598 ([MþNa]þ, 7%); HRLSI-MS calcd for C27H33NNaO6
-
1H, OH), 5.40 (tt, 1H, JH,H¼3.6 Hz, JH,H¼12.6 Hz, H-10), 4.12 (dd, 1H,
J9a,10¼1.8 Hz, J9a,9b¼12.3 Hz, H-9a), 3.88 (dd, 1H, J9b,10¼3.0 Hz, H-
10), 3.82 (d, 1H, H-6), 3.81 (dd, 1H, H-9b), 2.95 (m, 2H, 2OH),
2.26e2.14 (m, 2H, 2CHcyclohex.), 1.83e1.78 (m, 2H, 2CHcyclohex.), 1.63
SeSi ([MþNa]þ): 598.1140, found: 598.1126.
5.3. General procedure for the preparation of
spiroselenohydantoins 26 and 27
(m, 3H, 3CHcyclohex.), 1.36e1.11 (m, 3H, 3CHcyclohex.);
b pyran: d 5.05
(br s, 1H, H-7), 4.17 (dd, 1H, J9a,10¼1.7 Hz, J9a,9b¼12.9 Hz, H-9a), 4.13
To a solution of aminoester 5 (100 mg, 0.21 mmol) in THF
(m, 1H, H-6), 3.99 (dd, 1H, J9b,10¼3.6 Hz, H-10), 3.63 (dd, 1H, H-9b);
(1.5 mL) was added p-tolyl or
a-naphthyl isoselenocyanates

4896
P. Merino-Montiel et al. / Tetrahedron 68 (2012) 4888e4898
(0.31 mmol), and the mixture was stirred at inert atmosphere and
in the darkness until disappearance of the starting material was
observed by TLC. Then, crude reaction was concentrated to dryness,
and the residue was purified by column chromatography, using the
eluant indicated in each case.
H-2), 4.56 (t, 1H, J4,5a¼J4,5b¼6.3 Hz, H-4), 4.41 (dd, 1H,
J5a,5b¼10.5 Hz, H-5a) 4.17 (dd,1H, H-5b), 3.78 (s, 3H, OMe),1.49, 1.39
(2s, 3H each, 2CH3), 1.06 (s, 9H, C(CH3)3); 13C NMR (75.5 MHz,
CDCl3):
d
165.0 (C]O), 148.0 (C-40), 135.7, 135.6, 133.2, 133.0, 130.2,
130.1, 130.0, 128.9, 128.4, 128.0, 125.9 (AreC), 120.6 (C-50), 115.9
(CMe2), 104.9 (C-1), 86.8 (C-4), 86.3 (C-2), 75.7 (C-3), 63.5 (C-5),
53.2 (OMe), 27.1 (CH3), 27.0 (C(CH3)3), 26.8 (CH3), 19.5 (C(CH3)3);
LSIMS m/z 636 ([MþNa]þ, 67%); HRLSI-MS calcd for
C34H39N3NaO6Si ([MþNa]þ): 636.2506, found: 636.2508.
5.3.1. (5R,6S,8S,9S)-6-tert-Butyldiphenylsilyloxymethyl-8,9-(dime-
thylmethylenedioxy)-3-p-methylphenyl-2-selenoxo-7-oxa-1,3-
diazaspiro[4.4]nonan-4-one (26). p-Tolyl isoselenocyanate (60 mg)
was used, and the reaction proceeded for 16 h. Crude reaction was
purified by column chromatography (9:1/4:1 hexaneeEtOAc) to
give selenohydantoin 26 as a foam: 102 mg, 75%; Rf 0.33 (4:1
5.4.2. 5-O-tert-Butyldiphenylsilyl-3-[40-(cyclohex-100-en-100-yl)-10H-
10,20,30-triazol-10-yl]-3-deoxy-1,2-O-isopropylidene-3-C-methox-
ycarbonyl-b-D-arabinofuranose (29). 1-Ethynylcyclohexene (50 mL,
hexaneeEtOAc); ½a D25
ꢃ
ꢁ52 (c 0.50, CH2Cl2); 1H NMR (300 MHz,
CDCl3):
d
7.66e7.60 (m, 4H, AreH), 7.41e7.31 (m, 6H, AreH), 7.20
0.43 mmol) was used, and the reaction proceeded for 22 h. Column
(m, 4H, AreH), 5.94 (d, 1H, J8,9¼3.6 Hz, H-8), 4.76 (d, 1H, H-9),
4.35e4.30 (m, 2H, H-6, H-10a), 3.86 (m, 1H, H-10b), 2.39 (s, 3H,
PhCH3),1.49,1.31 (2s, 3H each, 2CH3), 0.99 (s, 9H, C(CH3)3); 13C NMR
chromatography (9:1 hexaneeEtOAc) afforded triazole 29: 88 mg,
67%; Rf 0.50 (4:1 hexaneeEtOAc); ½a D28
ꢃ
ꢁ12 (c 1.08, CH2Cl2); 1H
NMR (300 MHz, CDCl3): d
7.62e7.60 (m, 5H, AreH, H-50), 7.45e7.33
(75.5 MHz, CDCl3):
d
139.8, 135.7, 133.2, 133.1, 130.4, 130.2, 129.9,
(m, 6H, AreH), 6.45 (m, 1H, H-200), 5.91 (d, 1H, J1,2¼3.6 Hz, H-1), 5.73
(d, 1H, H-2), 4.52 (t, 1H, J4,5a¼J4,5b¼6.3 Hz, H-4), 4.36 (dd, 1H,
J5a,5b¼10.5 Hz, H-5a), 4.14 (dd, 1H, H-5b), 3.73 (s, 3H, OMe),
2.23e2.16 (m, 4H, 2CH2), 1.75e1.64 (m, 4H, 2CH2), 1.44, 1.36 (2s, 3H
each, 2CH3), 1.06 (s, 9H, C(CH3)3); 13C NMR (75.5 MHz, CDCl3):
127.9 (ꢄ3), 127.7 (AreC), 115.8 (CMe2), 105.5 (C-8), 87.5 (C-9), 84.8
(C-6), 62.2 (C-10), 27.2 (CH3), 27.1 (C(CH3)3), 26.9 (CH3), 21.4
(PhCH3), 19.3 (C(CH3)3); LSIMS m/z 673 ([MþNa]þ, 21%); HRLSI-MS
calcd for C33H38N2NaO850SeSi ([MþNa]þ): 673.1613, found:
673.1613.
d
165.0 (C]O), 149.7 (C-40), 135.7, 135.6, 133.2, 133.1, 123.0, 129.2,
127.9 (ꢄ2), 127.0 (Ar-C, C-100), 125.8 (C-200), 118.9 (C-50), 115.6
(CMe2), 104.9 (C-1), 87.1 (C-4), 86.0 (C-2), 75.5 (C-3), 63.6 (C-5), 53.1
(OMe), 27.0 (C(CH3)3), 26.9, 26.7 (2CH3), 26.3, 25.4, 22.5, 22.3
(4CH2), 19.4 (C(CH3)3); LSIMS m/z 640 ([MþNa]þ, 16%); HRLSI-MS
calcd for C34H43N3NaO6Si ([MþNa]þ): 640.2819, found: 640.2842.
5.3.2. (5R,6S,8S,9S)-6-tert-Butyldiphenylsilyloxymethyl-8,9-(dime-
thylmethylenedioxy)-3-
a
-naphthyl-2-selenoxo-7-oxa-1,3-diazaspiro
[4.4]nonan-4-one (27).
a-Naphthyl isoselenocyanate (73 mg) was
used, and the reaction proceeded for 40 h. Crude reaction was
purified by column chromatography (9:1/4:1 hexaneeEtOAc) to
give selenohydantoin 27 as a foam: 138 mg, 96%; Rf 0.25 (4:1
5.4.3. 5-O-tert-Butyldiphenylsilyl-3-(40-cyclopentyl-10H-10,20,30-tri-
azol-10-yl)-3-deoxy-1,2-O-isopropylidene-3-C-methoxycarbonyl-
b-D-
arabinofuranose (30). Cyclopentylacetylene (49 mL, 0.42 mmol) was
used, and the reaction proceeded for 12 h. Column chromatography
hexaneeEtOAc); ½a D24
ꢃ
ꢁ29 (c 0.44, CH2Cl2); 1H NMR (300 MHz,
CDCl3):
d
10.23 (br s, 1H, NH), 8.01 (d, 1H, JH,H¼8.1 Hz, AreH), 7.91
(m, 1H, JH,H¼8.1 Hz, AreH), 7.68e7.21 (m, 15H, AreH), 5.87 (d, 1H,
J8,9¼3.9 Hz, H-8), 4.97 (d, 1H, H-9), 4.45e4.35 (m, 2H, H-6, H-10a),
(4:1 hexaneeEtOAc) afforded triazole 30: 108 mg, 86%; Rf 0.34 (4:1
3.90 (dd, 1H, J6,1 b¼3.0 Hz, J1 a,1 b¼9.2 Hz, H-10b), 1.38, 1.30 (2s, 3H
hexaneeEtOAc); ½a D24
ꢃ
ꢁ7 (c 0.98, CH2Cl2); 1H NMR (300 MHz,
0
0
0
each, 2CH3), 0.97 (s, 9H, C(CH3)3); 13C NMR (75.5 MHz, CDCl3):
CDCl3): d
7.62e7.60 (m, 4H, AreH), 7.55 (s, 1H, H-50), 7.51e7.33 (m,
d
184.0 (C-2), 166.7 (C-4), 135.7, 134.4, 133.0, 132.8, 130.8, 130.2,
6H, AreH), 5.91 (d, 1H, J1,2¼3.6 Hz, H-1), 5.69 (d, 1H, H-2), 4.55 (t,
130.1, 130.0, 129.9, 128.6, 128.0, 127.9, 127.7 (ꢄ2), 126.7, 125.4, 122.7
(AreC), 116.1 (CMe2), 105.0 (C-8), 87.0 (C-9), 84.3 (C-6), 71.7 (C-5),
62.4 (C-10), 27.0 (C(CH3)3), 27.0, 26.3 (2CH3), 19.3 (C(CH3)3); LSIMS
m/z 709 ([MþNa]þ, 17%); HRLSI-MS calcd for C36H38N2NaO850SeSi
([MþNa]þ): 709.1613, found: 709.1633.
1H, J4,5a¼J4,5b¼6.3 Hz, H-4), 4.34 (dd, 1H, J5a,5b¼10.5 Hz, H-5a), 4.12
00 00
00 00
(dd, 1H, H-5b), 3.74 (s, 3H, OMe), 3.10 (q, 1H, J1 ,2 ¼J1 ,5 ¼8.1 Hz, H-
100), 2.04e2.00 (m, 2H, CH2), 1.77e1.54 (m, 6H, 3CH2), 1.43, 1.36 (2s,
3H each, 2CH3), 1.05 (s, 9H, C(CH3)3); 13C NMR (75.5 MHz, CDCl3):
d
165.2 (C]O), 152.8 (C-40), 135.7, 135.6, 133.2, 133.1, 130.0, 129.9,
127.9 (ꢄ2), (AreC),120.2 (C-50), 115.5 (CMe2),104.9 (C-1), 87.0 (C-4),
86.1 (C-2), 75.5 (C-3), 63.6 (C-5), 53.0 (OMe), 36.8 (C-100), 33.2, 33.1
(2CH2), 27.0 (C(CH3)3), 26.9, 26.7 (2CH3), 25.3 (ꢄ2) (2CH2), 19.4
(C(CH3)3); CIMS m/z 606 ([MþH]þ, 60%); HRCI-MS calcd for
C33H44N3O6Si ([MþH]þ): 606.2999, found: 606.2978.
5.4. General procedure for the synthesis of triazoles 28e31
To a solution of azidoester 4 (110 mg, 0.21 mmol) in a 1:1
mixture of acetoneeH2O (8 mL) were added copper(II) sulfate
(14.4 mg, 0.09 mmol), sodium ascorbate (31.2 mg, 0.16 mmol) and
the corresponding alkyne. The mixture was stirred at rt during the
time indicated in each case. Then, the mixture was concentrated to
dryness, and the residue was dissolved in CH2Cl2 (15 mL) and
washed with water (15 mL). The aqueous phase was extracted
with CH2Cl2 (2ꢄ50 mL); the combined organic fractions were
dried over MgSO4, filtered and the filtrate was concentrated to
dryness. The residue was purified by column chromatography,
using the eluant indicated in each case to give title triazoles as
foams.
5.4.4. 5-O-tert-Butyldiphenylsilyl-3-(40-n-butyl-10H-10,20,30-triazol-
10-yl)-3-deoxy-1,2-O-isopropylidene-3-C-methoxycarbonyl-
b
-
D
-ara-
binofuranose (31). Hex-1-yne (48
mL, 0.42 mmol) was used, and the
reaction proceeded for 12 h. Column chromatography (9:1 hex-
aneeEtOAc) afforded triazole 31: 97 mg, 76%; Rf 0.52 (4:1 hex-
aneeEtOAc); ½a 2D3
ꢃ
ꢁ7 (c 1.14, CH2Cl2); 1H NMR (300 MHz, CDCl3):
d
7.64e7.60 (m, 4H, AreH), 7.52 (s, 1H, H-50), 7.45e7.34 (m, 6H,
AreH), 5.91 (d, 1H, J1,2¼3.6 Hz, H-1), 5.71 (d, 1H, H-2), 4.54 (t, 1H,
J4,5a¼J4,5b¼6.3 Hz, H-4), 4.35 (dd, 1H, J5a,5b¼10.5 Hz, H-5a), 4.13 (dd,
00
00 00
1H, H-5b), 3.73 (s, 3H, OMe), 2.65 (t, 2H, J1 ,2 ¼7.6 Hz, H-1 ),1.58 (m,
5.4.1. 5-O-tert-Butyldiphenylsilyl-3-deoxy-1,2-O-isopropylidene-3-
C-methoxycarbonyl-3-(40-phenyl-10H-10,20,30-triazol-10-yl)-
b-D
binofuranose (28). Phenylacetylene (40
2H, H-200), 1.43, 1.34 (2s, 3H each, 2CH3), 1.41e1.26 (m, 2H, H-300),
1.05 (s, 9H, C(CH3)3), 0.91 (t, 3H, J3 ,4 ¼7.3 Hz, H-4 ); 13C NMR
00
00 00
-ara-
L, 0.36 mmol) was used,
m
(75.5 MHz, CDCl3): d
165.1 (C]O), 148.6 (C-40), 135.7, 135.6, 133.1,
and the reaction proceeded during 48 h. Column chromatography
130.0, 129.9, 127.9 (ꢄ2) (AreC), 121.3 (C-50), 115.6 (CMe2), 104.9 (C-
1), 87.0 (C-4), 86.0 (C-2), 75.4 (C-3), 63.5 (C-5), 53.0 (OMe), 31.4 (C-
100), 26.9 (C(CH3)3), 26.9, 26.7 (2CH3), 25.4 (C-200), 22.5 (C-300), 19.4
(C(CH3)3), 13.9 (C-400); CIMS m/z 594 ([MþH]þ, 12%); HRCI-MS calcd
for C32H44N3O6Si ([MþH]þ): 594.2999, found: 594.3003; Anal.
(19:1/9:1 hexaneeEtOAc) afforded triazole 28: 80 mg, 62%; Rf
0.44 (4:1 hexaneeEtOAc); ½a D28
ꢃ
ꢁ13 (c 1.04, CH2Cl2); 1H NMR
(300 MHz, CDCl3):
d
8.02 (s, 1H, H-50), 7.67e7.59 (m, 6H, AreH),
7.45e7.29 (m, 9H, AreH), 5.96 (d, 1H, J1,2¼3.6 Hz, H-1), 5.81 (d, 1H,

P. Merino-Montiel et al. / Tetrahedron 68 (2012) 4888e4898
4897
Calcd for C32H43N3O6Si: C, 64.73; H, 7.30; N, 7.08, found: C, 64.78; H,
7.39; N, 7.06.
J4,5a¼5.8 Hz, J4,5bz0 Hz, H-4), 4.85 (dd, 1H, H-2), 4.68 (dd, 1H,
J5a,5b¼10.4 Hz, H-5a), 4.40 (d, 1H, H-5b), 3.18e3.10 (m, 1H, H-100),
2.08e1.95 (m, 2H, CH2), 1.71e1.56 (m, 6H, 3CH2); 13C NMR
5.5. General method for the deprotection of triazoles 28e31
(75.5 MHz, DMSO-d6): a anomer: d
169.6 (C]O), 151.3 (C-40), 121.1
(C-50), 104.2 (C-1), 81.8 (C-4), 79.9 (C-2), 76.6 (C-3), 71.5 (C-5), 36.1
A solution of triazoles 28e31 (0.14 mmol) in a 9:1:2 mixture of
TFAeH2OeTHF (12 mL) was stirred at 50 ꢀC for 20 h. Then, the
solvent was removed under reduced pressure and co-evaporated
with toluene several times. The residue was purified as indicated
in each case to give triazolyl lactones 32e35 as anomeric mixtures.
(C-100), 32.7 (ꢄ2) (2CH2), 24.7 (2CH2);
b anomer: d 169.8 (C]O),
151.5 (C-40), 120.1 (C-50), 95.9 (C-1), 79.7 (C-4), 77.6 (C-2), 73.4 (C-5),
70.0 (C-3), 36.1 (C-100), 32.7 (ꢄ2) (2CH2), 24.7 (2CH2); CIMS m/z 296
([MþH]þ, 50%); HRCI-MS calcd for C13H18N3O5 ([MþH]þ): 296.1246,
found: 296.1247.
5.5.1. 3-C-3,5-Carbolactono-3-deoxy-3-(40-phenyl-10H-10,20,30-tri-
5.7.4. 3-(40-n-Butyl-10H-10,20,30-triazol-10-yl)-3-C-3,5-carbolactono-
azol-10-yl)-
D
-arabinofuranose (32). Triazole 28 was used. Crude
3-deoxy-D-arabinofuranose (35). Triazole 31 was used. Crude re-
reaction was washed with hexane (3ꢄ10 mL) to give triazole 32 as
action was purified by gel permeation chromatography (Biogel P2)
to give triazole 35 as a foam: 14 mg (36%); Rf 0.16 (5:1
a foam: 36 mg (86%); Rf 0.14 (5:1 CH2Cl2eMeOH); ½a D30
ꢃ
e92 (c 1.38,
8.79 (s,1H, H-
(CH3)2CO); 1H NMR (300 MHz, (CD3)2CO)
a
anomer:
d
CH2Cl2eMeOH); ½a D23
ꢃ
e180 (c 0.93, (CH3)2CO); 1H NMR (300 MHz,
d
50), 7.96 (m, 2H, AreH), 7.48e7.42 (m, 2H, AreH), 7.38e7.32 (m, 1H,
AreH), 5.85 (dd, 1H, J4,5a¼6.3 Hz, J4,5b¼2.7 Hz, H-4), 5.60 (s, 1H,
(CD3)2CO):
a
anomer:
8.07 (s, 1H, H-50), 5.74 (dd, 1H, J4,5a¼6.3 Hz,
J4,5b¼2.7 Hz, H-4), 5.56 (s, 1H, J1,2z0 Hz, H-1), 5.03 (s, 1H, H-2), 4.79
J
1,2z0 Hz, H-1), 5.16 (s, 1H, H-2), 4.85 (dd, 1H, J5a,5b¼10.2 Hz, H-5a),
(dd, 1H, J5a,5b¼10.2 Hz, H-5a), 4.41 (dd, 1H, H-5b), 2.72 (t, 2H,
00
00
8.74 (s, 1H, H-50), 7.96 (m, 2H,
J1 ,2 ¼7.5 Hz, H-1 ), 1.71e1.60 (m0,0 2H, H-3 ), 1.45e1.32 (m, 2H, H-
00 00
4.45 (dd, 1H, H-5b);
b
anomer:
d
200), 0.93 (t, 2H, J3 ,4 ¼7.2 Hz, H-4 );
b anomer: d
8.06 (s, 1H, H-50),
00 00
AreH), 7.48e7.42 (m, 2H, AreH), 7.38e7.32 (m, 1H, AreH), 5.56 (d,
1H, J1,2¼3.3 Hz, H-1), 5.52 (d, 1H, J4,5a¼5.1 Hz, J4,5bz0 Hz, H-4), 5.05
(d, 1H, H-2), 4.83 (dd, 1H, J5a,5b¼10.5 Hz, H-5a), 4.57 (d, 1H, H-5b);
5.52 (d, 1H, J1,2¼4.2 Hz, H-1), 5.46 (dd, 1H, J4,5a¼5.4 Hz, J4,5b¼0.9 Hz,
H-4), 4.89 (d, 1H, H-2), 4.79 (dd, 1H, J5a,5b¼10.5 Hz, H-5a), 4.72 (dd,
13C NMR (75.5 MHz, (CD3)2CO):
126.4, 126.3 (AreC); a anomer: d
d
131.6, 131.5, 129.7, 129.0, 128.9,
170.1 (C]O), 148.1 (C-40), 120.8 (C-
50), 105.6 (C-1), 83.1 (C-4), 82.1 (C-2), 77.9 (C-3), 72.6 (C-5);
1H, H-5b), 2.70 (t, 2H, J1 ,2 ¼7.5 Hz, H-1 ), 1.71e1.60 (m, 2H, H-2 ),
00
00
00 00
1.45e1.32 (m, 2H, H-300), 0.92 (t, 2H, J3 ,4 ¼7.2 Hz, H-4 ); 13C NMR
00
00 00
(75.5 MHz, (CD3)2CO):
a anomer: d
170.1 (C]O), 148.6 (C-40), 122.2
b
anomer:
d
171.2 (C]O), 148.3 (C-40), 121.7 (C-50), 96.7 (C-1), 80.9
(C-50), 105.6 (C-1), 83.3 (C-4), 82.2 (C-2), 77.5 (C-3), 72.4 (C-5), 32.2
(C-100), 25.8 (CH2), 22.8 (CH2), 14.0 (CH3);
anomer: 171.2 (C]O),
(C-4), 79.4 (C-2), 74.8 (C-5), 70.5 (C-3); LSIMS m/z 304 ([MþH]þ,
3%); 326 ([MþNa]þ, 6%); HRLSI-MS calcd for C14H14N3O5 ([MþH]þ):
304.0933, found: 304.0923.
b
d
148.9 (C-40), 122.4 (C-50), 96.5 (C-1), 80.8 (C-4), 79.6 (C-2), 74.7 (C-
5), 69.9 (C-3), 32.2 (C-100), 25.8 (CH2), 22.8 (CH2), 14.0 (CH3); CIMS
m/z 284 ([MþH]þ, 28%); HRCI-MS calcd for C12H18N3O5 ([MþH]þ):
284.1246, found: 284.1252; Anal. Calcd for C12H17N3O5: C, 50.88; H,
6.05; N, 14.83, found: C, 50.61; H, 6.02; N, 15.01.
5.5.2. 3-C-3,5-Carbolactono-3-[40-(cyclohex-100-en-100-yl)-10H-
10,20,30-triazol-10-yl]-3-deoxy-
-arabinofuranose (33). Triazole 29
D
was used. Crude reaction was purified by gel permeation chroma-
tography (Biogel P2) to give triazole 33 as a foam: 20 mg (50%); Rf
5.6. Enzymatic inhibition
0.15 (5:1 CH2Cl2eMeOH); ½a D24
ꢃ
e148 (c 0.80, (CH3)2CO); 1H NMR
(300 MHz, (CD3)2CO):
a
anomer:
d
8.25 (s, 1H, H-50), 6.56e6.49 (m,
The enzymatic assays were carried out as described pre-
viously.42 Each glycosidase assay was performed by preparing ten
2-mL samples in PS cuvettes containing 0.1 M phosphate buffer (pH
1H, H-200), 5.77 (dd, 1H, J4,5a¼6.0 Hz, J4,5b¼2.7 Hz, H-4), 5.56 (s, 1H,
J
1,2z0 Hz, H-1), 5.07 (s, 1H, H-2), 4.80 (dd, 1H, J5a,5b¼10.2 Hz, H-5a),
4.41 (dd, 1H, H-5b), 2.43e2.37 (m, 2H, H-600), 2.22e2.16 (m, 2H, H-
6.8) and substrate solution (p-nitrophenyl-
nitrophenyl- -glucopyranoside, p-nitrophenyl-
anoside, or o-nitrophenyl- -galactopyranoside). The concentra-
a-
D
-glucopyranoside, p-
300), 1.80e1.71 (m, 2H, H-500), 1.69e1.62 (m, 2H, H-400);
b
anomer:
b-
D
a-D-galactopyr-
d
8.19 (s, 1H, H-50), 6.56e6.49 (m, 1H, H-200), 5.52 (d, 1H, J1,2¼3.9 Hz,
b-D
H-1), 5.46 (dd, 1H, J4,5a¼5.4 Hz, J4,5b¼0.9 Hz, H-4), 4.93 (d, 1H, H-2),
4.79 (dd, 1H, J5a,5b¼10.5 Hz, H-5a), 4.52 (dd, 1H, H-5b), 2.43e2.37
(m, 2H, H-600), 2.22e2.16 (m, 2H, H-300), 1.80e1.71 (m, 2H, H-500),
tion of the substrate ranged from 0.25 to 4.0 Km. Water or inhibitor
solution plus water were also added up to a constant volume of
1.9 mL for the Km or the Ki measurement, respectively. Reaction was
started by adding 0.1 mL of dilute enzyme solution at 25 ꢀC and the
formation of the p- or o-nitrophenolate was monitored for 2 min by
measuring the increase of absorbance at 400 or 420 nm, re-
spectively. Initial rates were calculated from the slopes of each
reaction and were used to obtain two Hanes plots ([S]/V vs [S]), one
with and one without inhibitor. Inhibition constants (Ki) were
obtained from the formula Ki¼[I]/(Km0/Kmꢁ1), were [I] is the in-
1.69e1.62 (m, 2H, H-400); 13C NMR (75.5 MHz, (CD3)2CO):
a
anomer:
170.2 (C]O), 149.8 (C-40), 128.3 (C-100), 125.5 (C-200), 119.1 (C-50),
105.7 (C-1), 83.3 (C-4), 82.2 (C-2), 77.7 (C-3), 72.5 (C-5), 26.8 (C-600),
25.8 (C-300), 23.1 (C-500), 22.9 (C-400);
anomer: 171.1 (C]O), 150.0
d
b
d
(C-40), 128.2 (C-100), 125.2 (C-200), 120.1 (C-50), 96.7 (C-1), 80.8 (C-4),
79.6 (C-2), 74.7 (C-5), 70.2 (C-3), 26.8 (C-600), 25.8 (C-300), 23.1 (C-500),
22.9 (C-400); CIMS m/z 308 ([MþH]þ, 52%); HRCI-MS calcd for
C14H18N3O5 ([MþH]þ): 308.1246, found: 308.1245.
0
hibitor concentration in the cuvette and Km and Km are the enzy-
matic MichaeliseMenten constants in the absence and in the
presence of the inhibitor, respectively.
5.5.3. 3-C-3,5-Carbolactono-3-(40-cyclopentyl-10H-10,20,30-triazol-10-
yl)-3-deoxy-
D
-arabinofuranose (34). Triazole 30 was used. Crude
Glycogen phosphorylase activities were assayed in the direction
of glycogen synthesis43 by using commercial glycogen phosphor-
ylase b (rabbit muscle, 10 mg/mL) at 30 ꢀC in the presence of 1%
reaction was washed with Et2O (10 mL) and CH2Cl2 (10 mL) to give
triazole 34 as a foam: 28 mg (64%); Rf 0.15 (5:1 CH2Cl2eMeOH);
½
a 2D6
ꢃ
e128 (c 0.93, DMSO); 1H NMR (300 MHz, DMSO-d6):
oyster glycogen, a-D-glucose-1-phosphate, 1 mM AMP, with or
a
anomer:
d
8.25 (s, 1H, H-50), 6.83 (d, 1H, JOH,1¼3.7 Hz, OH-1), 6.57
without inhibitors at pH 6.8 (50 mM triethanolamine/HCl, 1 mM
dithiothreitol and 1 mM EDTA buffer). Pi concentration was de-
termined using the method developed by Taussky and Shorr,44
using as developer 0.18 mM FeSO4 and 0.05 M (NH4)MoO4 in the
presence of 0.5 M H2SO4 at 720 nm. Hanes plots were built using
the initial velocity from each reaction, as indicated above for
glycosidases.
(d, 1H, JOH,2¼5.0 Hz, OH-2), 5.56 (dd, 1H, J4,5a¼6.3 Hz, J4,5b¼2.6 Hz,
H-4), 5.37 (d, 1H, J1,2z0 Hz, H-1), 4.94 (d, 1H, H-2), 4.75 (dd, 1H,
J5a,5b¼10.1 Hz, H-5a), 4.33 (dd, 1H, H-5b), 3.18e3.10 (m, 1H, H-100),
2.08e1.95 (m, 2H, CH2), 1.71e1.56 (m, 6H, 3CH2);
b anomer: d 8.16
(s, 1H, H-50), 7.23 (d, 1H, JOH,1¼4.0 Hz, OH-1), 5.71 (d, 1H,
JOH,2¼8.0 Hz, OH-2), 5.33 (t, 1H, J1,2¼3.7 Hz, H-1), 5.24 (d, 1H,

4898
P. Merino-Montiel et al. / Tetrahedron 68 (2012) 4888e4898
ꢀ
ꢀ
ꢀ
12. Somsak, L.; Nagy, V.; Vidal, S.; Czifrak, K.; Berzsenyi, E.; Praly, J.-P. Bioorg. Med.
Chem. Lett. 2008, 18, 5680e5683.
5.7. Free radical scavenging
ꢀ
13. Somsak, L.; Czifrak, K.; Toth, M.; Bokor, E.; Chrysina, E. D.; Alexacou, K.-M.;
The free radical-scavenging activity was measured using the
DPPH (1,1-diphenyl-2-picrylhydrazyl radical) method45 in a UV
spectrometer using PS cuvettes.
Hayes, J. M.; Tiraidis, C.; Lazoura, E.; Leonidas, D. D.; Zographos, S. E.; Oiko-
nomakos, N. G. Curr. Med. Chem. 2008, 15, 2933e2983.
14. (a) Sridhar, P. R.; Seshadri, K.; Reddy, G. M. Chem. Commun. 2012, 756e758; (b)
Goyard, D.; Telligmann, S. M.; Goux-Henry, C.; Boysen, M. M. K.; Framery, E.;
Gueyrard, D.; Vidal, S. Tetrahedron Lett. 2010, 51, 374e377; (c) Soengas, R. G.
To a 60
was added 30
m
M solution of DPPH in HPLC-grade methanol (1.7 mL)
L of the antioxidant solution in MeOH (five different
ꢀ
ꢀ
Synlett 2010, 2549e2552; (d) Maza, S.; Lopez, O.; Martos, S.; Maya, I.;
Fernandez-Bolanos, J. G. Eur. J. Org. Chem. 2009, 5239e5246; (e) Fuentes, J.;
Salameh, B. A. B.; Pradera, M. A.; Fernandez de Cordoba, F. J.; Gasch, C. Tetra-
m
ꢀ
~
concentrations) or pure methanol (control). The corresponding
mixture was kept in the darkness for 30 min and then the absor-
bance at 515 nm was measured against a MeOH blank in order to
calculate the EC50 values, that is, the concentration of the antioxi-
ꢀ
hedron 2006, 62, 97e111.
15. (a) Kancharla, P. K.; Vankar, Y. D. J. Org. Chem. 2010, 75, 8457e8464; (b) Babu, B.
R.; Keinicke, L.; Petersen, M.; Nielsen, C.; Wengel, J. Org. Biomol. Chem. 2003, 1,
3514e3526.
dant (expressed in mM) required to reduce the concentration of the
16. (a) Suryawanshi, S. B.; Dushing, M. P.; Gonnade, R. G.; Ramana, C. V. Tetrahe-
dron 2010, 66, 6085e6096; (b) Richter, S. N.; Menegazzo, I.; Nadai, M.; Moroc,
S.; Palumboc, M. Org. Biomol. Chem. 2009, 7, 976e985; (c) Gasch, C.; Illangua, J.
M.; Merino-Montiel, P.; Fuentes, J. Tetrahedron 2009, 69, 4149e4155; (d)
Maurya, S. K.; Hotha, S. Tetrahedron Lett. 2006, 47, 3307e3310; (e) Van Nhien,
A. N.; Domínguez, L.; Tomassi, C.; Torres, M. R.; Len, C.; Postel, D.; Marco-
Contelles, J. Tetrahedron 2004, 60, 4709e4727; (f) Nguyen, A.; Villa, P.; Ronco,
G.; Postel, D. J. Pharm. Pharmacol. 2001, 53, 939e943; (g) Shibata, K.; Hiruma,
K.; Kanie, O.; Wong, C.-H. J. Org. Chem. 2000, 65, 2393e2398; (h) Postel, D.;
Nguyen Van Nhien, A.; Villa, P.; Ronco, G. Tetrahedron Lett. 2001, 42,
1499e1502.
DPPH to 50% of its initial value. All the measurements were carried
out in triplicate. The remaining DPPH concentration was calculated
using the expression:
Asample
%DPPH remaining ¼
ꢄ 100
Acontrol
where Asample and Acontrol refer to the absorbances at 515 nm of the
free radical in the sample and control solutions, respectively.
17. (a) Maity, J. K.; Ghosh, R.; Drew, M. G. B.; Achari, B.; Mandal, S. K. J. Org. Chem.
2008, 73, 4305e4308; (b) Roy, A.; Achari, B.; Mandal, S. B. Tetrahedron Lett.
2006, 47, 3875e3879.
Acknowledgements
ꢀ
ꢀ
ꢀ
~
18. Gasch, C.; Merino-Montiel, P.; Lopez, O.; Fernandez-Bolanos, J. G.; Fuentes, J.
Tetrahedron 2010, 66, 9964e9973.
19. Sharma, G. V. M.; Reddy, V. G.; Krishna, P. R.; Sankar, A. R.; Kunwar, A. C. Tet-
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5284e5294.
ꢀ
ꢀ
We thank the Direccion General de Investigacion of Spain and
the Junta de Andalucía (grants numbers CTQ2008-02813 and
FQM134). P.M.-M. thanks the CONACYT of Mexico and the
ꢀ
Fundacion Carolina of Spain for the award of a fellowship.
Supplementary data
24. Cesare, V.; Lyons, T. M.; Lengyel, I. Synlett 2002, 1716e1720.
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Supplementary data related to this article can be found online at
doi:10.1016/j.tet.2012.03.087.
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