Spiranic d-gluco-configured N-substituted thiohydantoins as potential enzymatic inhibitors

Article abstract of DOI:10.1016/j.tet.2010.09.109

We have explored the preparation of conformationally-restricted pseudonucleosides bearing a spiranic thiohydantoin scaffold on C-3 of the sugar moiety by coupling partially protected 3-amino-3-methoxycarbonyl and 3-isothiocyanato-3-methoxycarbonyl glucofu

Full text of DOI:10.1016/j.tet.2010.09.109

Tetrahedron 66 (2010) 9964e9973
Contents lists available at ScienceDirect
Tetrahedron
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Spiranic
D-gluco-configured N-substituted thiohydantoins as potential enzymatic
inhibitors
ꢀ
*
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
~
ꢀ
Consolacion Gasch , Penelope Merino-Montiel, Oscar Lopez, Jose G. Fernandez-Bolanos, Jose Fuentes
ꢀ
Departamento de Química Organica, Facultad de Química, Universidad de Sevilla, Apartado 1203, E-41071 Seville, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 July 2010
Received in revised form 3 September 2010
Accepted 15 September 2010
Available online 19 October 2010
We have explored the preparation of conformationally-restricted pseudonucleosides bearing a spiranic
thiohydantoin scaffold on C-3 of the sugar moiety by coupling partially protected 3-amino-3-methox-
ycarbonyl and 3-isothiocyanato-3-methoxycarbonyl glucofuranose derivatives with alkyl(aryl)iso-
thiocyanates or with alkyl(aryl)amines; the key step is a spontaneous or thermal-induced intramolecular
nucleophilic substitution of transient thioureas. Upon deprotection, final spiranic thiohydantoins were
evaluated as glycosidase and glycogen phosphorylase inhibitors.
Keywords:
Pseudonucleosides
Thiohydantoins
Ó 2010 Elsevier Ltd. All rights reserved.
Spiranic compounds
Hydantocydin analogues
Glycosidase inhibitors
1. Introduction
The interesting biological properties exerted by hydantocidin
prompted researchers to carry out not only its total synthesis13 but
The preparation of nucleoside analogues is a challenging task in
Organic and Medicinal Chemistry, due to the important biological
activities exhibited by many of such derivatives, considered to be
potential therapeutic agents mainly as antiviral and anticancer
drugs.1 Among the plethora of derivatives reported in the literature,
deoxy nucleosides,2 acyclic3 and carbocyclic4 nucleosides, thio-,5
aza-6 and selenonucleosides,7 or derivatives bearing modified
heterocyclic scaffolds8 have emerged as new families with en-
hanced activities and improved bioavailability.9
A relatively novel family of nucleosides is comprised of spi-
ronucleosides, where carbohydrate derivatives and heterocyclic
moieties share a spiranic carbon atom.10 Such structure has gained
attention since the isolation in 1991 of (þ)-hydantocidin 1 from the
cultured broth of Streptomyces hygroscopicus;11 hydantocidin was
found to possess a potent herbicidal activity towards numerous
weeds, and to be a plant growth regulator, and at the same time, it
lacked toxicity against microorganisms, fishes and mammals.12
also to prepare analogues by modifying the sugar moiety,14 the
heterocyclic ring,15 or by modifying both units.16 Glycopyr-
anosylidene-spiro derivatives bearing hydantoin,17 thio-
hydantoin.17 or oxathiazole18 scaffolds were found to be among the
most potent inhibitors of glycogen phosphorylase, an enzyme that
catalyzes glycogen breakdown, and therefore a potential target in
the treatment of metabolic disorder diseases such as diabetes type
II.19 Some other spiranic derivatives structurally related to hydan-
tocidin turned out to be potent salivary
a
-amylase,20 or adenylo-
succinate synthetase inhibitors.21
In this context, we have recently reported a series of spiranic
pseudonucleosides bearing oxazolidine-, imidazolidine-2-thione
(selone), thiohydantoin-, perhydrooxazine- and azetidine-based
heterocycles.22
2. Results and discussion
In this communication we have explored the coupling of iso-
thiocyanates and amines with protected 3-amino-3-methoxy-
carbonyl
and
with
3-isothiocyanato-3-methoxycarbonyl
glucofuranose derivatives 3 and 4,22a respectively, to furnish con-
formationally-restricted pseudonucleosides 17e28 (Scheme 1),
where the spiranic annulation takes place at the C-3 of the sugar
residue. It has been claimed that reducing the conformational
flexibility in nucleosides and nucleotides23 might enable the opti-
mal puckering between the nucleoside or nucleotide and the
* Corresponding author. E-mail address: cgasch@us.es (C. Gasch).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.09.109

C. Gasch et al. / Tetrahedron 66 (2010) 9964e9973
9965
This procedure was successfully applied to the preparation of
N-substituted thiohydantoins bearing aryl (phenyl, naphthyl, p-tolyl)
and alkyl residues, including sugar moieties (indolylethyl, n-butyl,
cyclohexyl, per-O-acylated
b-D-glucopyranosyl, a-D-glucopyranos-2-
yl and -arabinopyranosyl) in moderate to almost quantitative
a-D
yields (Table 1). When per O-acetylated b-D-glucopyranosylamine
and glucosamine hydrobromides29,30 (Table 1, entries 7 and 8, re-
spectively) were used, anequivalentofEt3N wasadded tothereaction
mixture.
Furthermore, the use of diamines (Table 1, entries 10, 11) or tri-
amines (Table 1, entry 12) afforded dimeric thiohydantoins 26, 27
and trimeric derivative 28 in 88e97% yields, as examples of potential
multivalent ligands. Multivalency has been used to enhance the
potency of certain enzymatic inhibitors, especially against receptors
bearing multiple recognition sites; this effect is proposed to occur by
the increase of the local concentration of ligands, and it has also been
used to modulate the pharmacokinetic properties of the inhibitor.31
The formation of thiohydantoins 18e22, 26, and 28 proceeded
smoothly at rt, as the corresponding intermediate thioureas un-
derwent spontaneous cyclization to furnish title compounds. On the
other hand, for the synthesis of derivatives 17, 23e25, 27 higher
temperatures ranging from 40 ꢀC to 66 ꢀC (see Experimental) were
needed in order to favour the intramolecular cyclization in the thi-
ohydantoin ring formation. For thiohydantoin 24, besides higher
temperatures, basic catalysis was proved to be essential for the cy-
clization step. In some cases, the transient thiourea could be visu-
alized by TLC as a spot with a lower Rf than the final thiohydantoin.
Scheme 1. Preparation of protected 5-spirothiohydantoins 17e28.
interacting biomolecule, due to entropic factors;24 furthermore, it
has also been reported an increased stability of some constrained
nucleosides upon radical-induced degradation reactions.25
Intermediates 3 and 4 can be accessed starting from 1,2:5,6-di-O-
isopropylidene-a-D
-ribo-hexofuranos-3-ulose 2,26 by nucleophilic
addition of chloroform to the ketone promoted by LiHMDS (lithium
hexamethyldisilazide), followed by modified CoreyeLink reaction27
with NaN3, MeOH and DBU to furnish, via a transient dichloroepoxy
intermediate, the corresponding 3-azido-3-methoxycarbonyl de-
rivative28 in a complete stereoselective fashion. Reduction of the
azido group under catalytic hydrogenation afforded 3. Conversion of
3 into isothiocyanate derivative 4 was accomplished by treatment
with thiophosgene in a heterogeneous CaCO3/CH2Cl2/H2O system.22a
Treatment of aminoester 3 or isothiocyanato ester 4 with an
aryl/alkyl isothiocyanate (Table 1, entries 1 and 9) or with an aryl/
alkyl amine (Table 1, entries 2e8, 10e12), respectively, resulted in
the formation of intermediate thioureas 5e16, which either spon-
taneously or promoted by heating and/or base underwent an
intramolecular nucleophilic substitution on the carbonyl carbon
(Scheme 1) to give a thiohydantoin ring with a spiranic annulation
(compounds 17e28); such derivatives can be considered as struc-
tural analogues of natural (þ)-hydantocidin 1.
When
a-D-arabinopyranosyl isothiocyanate was coupled with
aminoester 3, thiourea 13 (Scheme 1) was isolated as the only com-
pound even when reaction was heated up to 60 ꢀC; the structure was
supported by 1H NMR and 13C NMR spectra, which showed the
presence of the methoxycarbonyl group (3.81 and 53.0 ppm), to-
gether with two NH peaks in 1H NMR spectrum (7.97, 7.31 ppm),
which thus evidence the existence of a non-spiranic structure. Reso-
nance of the thiocarbonyl group (184.8 ppm, Table 2) was also con-
sistent with the thiourea moiety. Furthermore, the arabinopyranosyl
residue was found to be in the 1C4 conformation, as deduced from the
Table 1
Synthesis of protected
D-gluco-configured 5-spirothiohydantoins
Entry
Amine
Isothiocyanate
Thiohydantoin
Compound
Yielda (%)
1
3
17
68
2
4
18
50
3
4
19
80
(continued on next page)

9966
C. Gasch et al. / Tetrahedron 66 (2010) 9964e9973
Table 1 (continued)
Entry
Amine
Isothiocyanate
Thiohydantoin
Compound
Yielda (%)
4
4
4
4
20
79
5
21
88
6
22
94
7
4
23
76
8
4
24
23
9
3
25
93b
10
4
26
93

C. Gasch et al. / Tetrahedron 66 (2010) 9964e9973
9967
Table 1 (continued)
Entry
Amine
Isothiocyanate
Thiohydantoin
Compound
Yielda (%)
11
4
27
88
12
4
28
97
a
Isolated yields.
Starting from intermediate thiourea 13.
b
Table 2
Selected NMR data for compounds 3,4,13,17e29
Compd
H-6
H-9
C-2
C-5
C-6
C-9
322a,a
422a,a
13a
4.67 (H-4)b
4.60 (H-4)b
4.60 (H-4)b
4.42
4.26 (H-2)b
4.72 (H-2)b
4.82 (H-20)b
4.77
d
68.2 (C-3)b
75.0 (C-3)b
74.2 or 74.3 (C-30)b
71.7
82.0 (C-4)b
87.7 (C-4)b
80.8 (C-40)b
82.9
88.7 (C-2)b
82.0 (C-2)b
88.4 (C-20)b
85.7
142.5 (C]S)b
184.8 (C]S)b
183.7
17a
18a
4.47
4.80
183.9
72.2
81.7
86.5
4.46c
4.91c
184.2c
72.0c
82.7c
86.0c
19a
20a
21a
22a
23a,d
24a
4.41
4.42
4.36
4.32
4.77
4.55
4.63
4.62
184.0
183.5
183.9
184.2
182.7, 180.6
183.5
71.7
71.1
71.1
70.0
71.6, 70.4
71.0
82.9
82.3
82.3
82.5
82.5, 79.5
80.5
85.7
85.5
85.9
85.8
86.6, 86.2
88.0
4.37
4.62
4.39
4.53
ee
4.67c
25a
26a
27a
28a
29f
4.41
4.35
4.38e4.35
4.35
4.30
4.61
4.64
4.63
4.70
182.7
183.7
183.9
183.3
184.5
70.0
71.1
71.1
71.1
70.9
82.4
82.3
82.3
82.2
83.3
85.6
85.8
85.9
85.8
86.8
4.68
a
In CDCl3.
In brackets, sugar numbering.
Minor atropisomer.
A roughly 1:1 mixture of atropisomers.
Not observed.
In CD3OD.
b
c
d
e
f
high J1,2, J2,3 coupling constants (9.6 Hz), together with the low value
for the J3,4 coupling constant (3.3 Hz). These data are in accordance
with reported data for per-O-acylated arabinosyl thioureas.32 Only
when thiourea 13 was refluxed in THF, the corresponding N-a-D-
arabinopyranosyl-thiohydantoin 25 (Table 1, entry 9) was obtained in
excellent yield (93%) after chromatographic purification.
NMR spectra at rt. For derivative 18, bearing a naphthyl residue, the
ratio of the major atropisomer decreased upon heating in DMSO-d6
from rt to 90 ꢀC from 4.1:1 to 2.9:1, as depicted in Table 3, although
no coalescence was reached. The maximum energy level must be
found when the aromatic ring is coplanar with the thiohydantoin
ring, due to steric hindrance between the naphthyl moiety and the
carbonyl and thiocarbonyl groups. On the other hand, the local
minima for these two atropisomers must be found when the aro-
matic system is in a perpendicular orientation with respect to the
Thiohydantoins 17e28 lacked the signal for the methoxycarbonyl
group in 1H and 13C NMR spectra, and only one NH group was ob-
served ranging from 7.26 to 9.58 ppm. Compounds 17e28 exhibited
a shielding of proton H-6 of roughly 0.2e0.3 ppm when compared
thiohydantoin ring. N-b-D-glucopyranosyl and N-a-D-glucopyr-
anos-2-yl thiohydantoins derivatives 23 and 24 also showed
with parent
depicted in Table 2. Moreover, resonances of spiranic carbon C-5
were deshielded as compared with -amino ester 3 (ca. 2e4 ppm),
a-amino ester 3 and
a
-isothiocyanato ester 4,22a as
Table 3
a
Atropisomeric mixtures for compound 18
and shielded with respect to isothiocyanato ester 4 (ca. 3e5 ppm);
these data are in agreement with the observation that an adjacent
carbon to an NCS group is deshielded.22a
Free rotation around the NeR bond was restricted when bulky
substituents were present; thus, for thiohydantoins 18, 23 and 24,
two families of atropisomeric compounds were detected in their
Solvent
Temperature
Ratio
CDCl3
rt
rt
60 ꢀC
90 ꢀC
3.8:1
4.1:1
4.0:1
2.9:1
DMSO-d6
DMSO-d6
DMSO-d6

9968
C. Gasch et al. / Tetrahedron 66 (2010) 9964e9973
atropisomerism, the rotamers being in a ratio roughly 1:1 and a 6:1,
respectively in CDCl3 at rt. In these cases, the presence of a per-O-
acylated sugar moiety prevented the free rotation around the NeR
bond. In thiohydantoin 24, changing the solvent to DMSO-d6 led to
only one rotamer in the sample.
It is noteworthy that when the glucopyranosyl moiety of 23 was
partially deprotected using methanolic NaOMe (Scheme 2), no
atropisomerism was observed for the corresponding O-deacylated
derivative 29; it is therefore clear that in compound 23 the pres-
ence of the acetoxy groups prevents free rotation and thus leads to
the presence of two atropisomeric compounds, observable at rt.
(Table 5). The pyranoid structure was evidenced by the high cou-
pling constants J6,7 (7.8 Hz-8.1 Hz) and J9,10 (9.5e10.2 Hz), thus in-
dicating a trans-arrangement of such protons.
Table 5
Anomeric ratios for thiohydantoins 30e35
Compd
30
31
32
33
34
35
a
/b
ratio
1:9.9a
1:3.5b
1:2.9b
1:10a
1:25b
1:19a
1:5b
1:2.9c
1:4c
a
b
c
D2O.
CD3OD.
DMSO-d6.
Furthermore, resonances of the adjacent carbons to the thio-
hydantoin scaffold (C-10 and C-6 for the pyranoid structures) are
strongly shielded for derivatives 30e35 (Table 4) in comparison
with these data for protected furanoid counterparts (C-6, C-9). For
example, C-10 showed a resonance in the range 68.7e70.3 ppm for
thiohydantoins 30e35, whereas the same carbon (C-6) resonated in
the range 83.3e81.7 ppm for the corresponding furanoid de-
rivatives (Table 2). These observations are again indicative of the
final compounds exhibiting a pyranoid structure, and are also ful-
Scheme 2. O-deacetylation of glucopyranosyl thiohydantoin 23.
filled for the corresponding minor
Unprotected thiohydantoins 30e33 and 35 were assayed as
glycosidase inhibitors ( -glucosidase, -glucosidase, -galactosi-
dase) and glycogen phosphorylase b inhibitors (Table 6). None of
the tested compound showed inhibition against glycogen phos-
phorylase. Of the simple thiohydantoins, only compound 30,
bearing an aromatic residue on N-3, showed a weak inhibition (8%
a-anomers.
Compounds 17, 21, 22, 26e28 were deprotected under acidic
conditions (9:1 TFA/H2O), as depicted in Scheme 3 to afford the
corresponding spiranic thiohydantoins 30e35 in almost quantita-
tive yields; trimeric thiohydantoin 35 was isolated as its tri-
fluoroacetate salt.
a
b
b
at 1 mM concentration) against
b-glucosidase (almonds) and b-
galactosidase (Aspergillus oryzae). A similar inhibition was found for
dimeric thiohydantoin 33 against a-glucosidase (baker’s yeast) and
Table 6
Inhibition activities of compounds 30e33, 35a
Compound
30
N.I.b
31 32 33 35
N.I. N.I. 7% N.I.
a-Glucosidase (baker’s yeast)
b-Glucosidase (almonds)
b-Galactosidase (A. oryzae)
b-Galactosidase (E. coli)
8% (4.07) N.I. N.I. 6% 26% (1.74)
8%
N.I.
N.I. N.I. N.I. N.I.
25% N.I. N.I. 25%
N.I. N.I. N.I. N.I.
Scheme 3. Deprotection of thiohydantoins.
Glycogen phosphorylase b (rabbit muscle) N.I.
NMR data of compounds 30e35 suggested that they are in the
pyranosic form (Table 4) and as solvent-dependent anomeric
a
In brackets Ki values (mM).
No inhibition at 1 mM concentration.
b
equilibrium, where the
b-anomers are present in a higher ratio
Table 4
Selected NMR data for compounds 30e35
Compd
H-6
H-9
H-10
J6,7 (Hz)
C-5
C-9
C-10
30a
3.47b
3.88c
3.24b
dc,d
4.17b
3.75b
8.1b
4.7c
7.8b
3.2c
8.0b
4.0c
8.1b
4.5c
8.0b
4.5c
8.1b
4.8c
74.8 or 74.7b,e
74.8 or 74.7b,e
70.3b
dc,d
dc,d
dc,d
dc,d
dc,d
31f
32f
33a
34a
35g
3.90b
3.91e3.85c
3.90b
3.86c
3.47b
dc,d
72.2b
73.5b
68.2b
67.3c
67.5b
67.5c
68.7b
dc,d
74.1c
67.9c
3.22b
3.65e3.02c
3.56b
4.02c
3.37b
dc,d
3.45b
3.55c
3.83b
dc,d
71.3b
73.4b
73.1c
67.7c
4.23b
4.26c
73.6 or 73.7b,e
dc,d
73.7 or 73.6b,e
dc,d
4.17b
4.13c,d
4.21 b
dc,d
3.51b
74.3b
74.8b
70.1b
dc,d
dc,d
dc,d
dc,d
3.57b
3.84b
dc,d
73.0, 73.6b,e
73.6 or 73.0b,e
68.1b
dc,d
4.00c
dc,d
dc,d
a
In CD3OD.
b
c
d
e
f
b
-anomer.
-Anomer.
Not observed.
a
Unambiguous assignment not possible.
In DMSO-d6.
In D2O.
g

C. Gasch et al. / Tetrahedron 66 (2010) 9964e9973
9969
b
-glucosidase (almonds). Although quite moderate, multivalent
thiohydantoin 35, exhibiting a symmetrical trimeric structure, was
found to be a weak inhibitor of -glucosidase and of -galactosidase
thiohydantoin 28) and the solution was kept stirring at x ꢀC for y h,
until TLC showed disappearance of the starting material. The sol-
vent was then removed under reduced pressure and the residue
was purified by column chromatography using the eluant in-
dicated in each case.
b
b
(Escherichia coli) (26 and 25%, respectively at 1 mM concentration).
Method B: To a solution of isothiocyanate 4 (100 mg, 0.28 mmol)
in THF (1.5 mL) was added the corresponding amine (0.42 mmol)
and triethylamine (0.46 mmol). The solution was kept stirring at
3. Conclusions
We have explored the high-yielding preparation of pseudonu-
x
ꢀC for y h, until TLC showed disappearance of the starting mate-
cleosides of
D-gluco configuration and a locked conformation
rial. The solvent was then removed under reduced pressure and the
residue was purified by column chromatography using the eluant
indicated in each case.
bearing spiro-annulated N-substituted thiohydantoins in the C-3
position of the sugar moiety; for that purpose we used an O-pro-
tected a-amino or a-isotiocyanate ester as the key intermediate,
which was coupled with alkyl(aryl) isothiocyanates or amines, in-
cluding sugar derivatives. This methodology gave access to furanoid
spiranic derivatives that were transformed into pyranoid scaffolds
upon acidic deprotection. Some unprotected derivatives were
found to be weak inhibitors of glycosidases.
4.2.1. (5S,6S,8R,9R,40R)-6-(20,20-Dimethyl-10,30-dioxolan-40-yl)-8,9-
(dimethylmethylenedioxy)-3-a-naphthyl-2-thioxo-7-oxa-1,3-dia-
zaspiro[4.4]nonan-4-one (18). Method A: x¼25 ꢀC, y¼24 h. Column
chromatography: 50:1 CH2Cl2/MeOH. Amorphous solid (65.8 mg,
26
50%). [
a
]
þ39 (c 1.15, CH2Cl2); 1H NMR (500 MHz, CDCl3): major
D
atropisomer
d 9.37 (s, 1H, NH), 8.03e7.99 (m, 2H, Ar), 7.94 (d, 1H,
4. Experimental section
4.1. General procedures
JH,H¼7.8 Hz, Ar), 7.60e7.46 (m, 4H, Ar), 5.93 (d, 1H, J8,9¼3.5 Hz, H-8),
0
4.80 (d, 1H, H-9), 4.47 (d, 1H, J6,4 ¼9.5 Hz, H-6), 4.38 (ddd, 1H,
J4 ,5 a¼5.5 Hz, J4 ,5 b¼3.0 Hz, H-40), 4.15e4.09 (m, 2H, H-50a, H-50b),
0
0
0
0
1.63, 1.45, 1.34, 1.21 (4s, 3H each, 4CH3); minor atropisomer:
d 9.32
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 homo-
nuclear COSYand heteronuclear 2D correlated spectra, respectively.
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 thioglycerol as matrix and
NaI as additive. TLC was performed on aluminium pre-coated
sheets (E. Merck Silica Gel 60 F254); spots were visualized by UV
light, by charring with 10% H2SO4 in EtOH or with 3% ninhydrin in
EtOH. Column chromatography was performed using E. Merck
(s, 1H, NH), 7.68 (d, 1H, JH,H¼8.0 Hz, Ar), 7.41 (d, 1H, JH,H¼7.2 Hz, Ar),
5.95 (d, 1H, J8,9¼3.6 Hz, H-8), 4.91 (d, 1H, H-9), 4.46 (d, 1H,
J6,4 ¼9.5 Hz, H-6), 4.29 (ddd, 1H, J4 ,5 a¼5.7 Hz, J4 ,5 b¼3.4 Hz, H-40),
0
0
0
0
0
4.15e4.12 (m, 1H, H-50a), 4.04 (dd, 1H, J5 a,5 b¼9.3 Hz, H-50b), 1.61,
0
0
1.40, 1.35, 1.26 (4s, 3H each, 4CH3); 13C NMR (125.7 MHz, CDCl3):
major atropisomer
d 183.9 (C-2), 168.0 (C-4), 134.5, 130.7, 130.6,
129.5, 128.6, 127.8, 126.9, 126.8, 125.5, 123.6 (Ar), 114.8, 111.0
(2CMe2), 105.3 (C-8), 86.5 (C-9), 81.7 (C-6), 73.3 (C-40), 72.2 (C-5),
67.2 (C-50), 27.0, 26.7, 26.6, 25.7 (4CH3); minor atropisomer:
d 184.2
(C-2), 167.9 (C-4), 134.4, 130.5, 130.2, 129.9, 128.8, 127.5, 127.3, 125.4,
122.2 (Ar), 114.8, 110.6 (CMe2), 105.8 (C-8), 86.0 (C-9), 82.7 (C-6),
73.5 (C-40), 72.0 (C-5), 67.4 (C-50), 27.0, 26.7 26.6, 25.6 (4CH3);
HRLSI-MS m/z calcd for C24H26N2NaO6S, [MþNa]þ: 493.1409,
found: 493.1418.
Silica Gel 60 (40e63 mm). Compound 2 was prepared according to
the described literature procedure.26 Compounds 3 and 4 were
4.2.2. (5S,6S,8R,9R,40R)-6-(20,20-Dimethyl-10,30-dioxolan-40yl)-8,9-
(dimethylmetilenedioxy)-2-thioxo-3-p-tolyl-7-oxa-1,3-diazaspiro
[4.4]nonan-4-one (19). Method A: x¼25 ꢀC, y¼24 h. Column chro-
matography: 50:1 CH2Cl2/MeOH. Amorphous solid (97 mg, 80%).
prepared as we described in a previous work.22a
4.1.1. (5S,6S,8R,9R,40R)-6-(20,20-Dimethyl-10,30-dioxolan-40-yl)-8,9-
(dimethylmethylenedioxy)-3-phenyl-2-thioxo-7-oxa-1,3-diazaspiro
[4.4]nonan-4-one (17). To a solution of aminoester 3 (63.8 mg,
[
a
]
23 þ65 (c 1.15, CH2Cl2); 1H NMR (500 MHz, CDCl3):
d 9.34 (s, 1H,
D
NH), 7.30e7.29, 7.20e7.18 (m, 4H, Ar), 5.96 (d, 1H, J8,9¼3.5 Hz, H-8),
0.20 mmol) in THF (1.5 mL) was added phenyl isothiocyanate (36 mL,
0
4.77 (d, 1H, H-9), 4.41 (d, 1H, J6,4 ¼9.5 Hz, H-6), 4.22 (ddd, 1H,
0.30 mmol, 1.5 equiv). The corresponding solution was stirred at rt
for 24 h and at 60 ꢀC for 16 h. Then, the solvent was removed under
reduced pressure and the residue was purified by column chroma-
tography (7:3 hexane/EtOAc) to give 17 as an amorphous solid
J4 ,5 a¼6.0 Hz, J4 ,5 b¼4.0 Hz, H-40), 4.12 (dd, 1H, J5 a,5 b¼9.0 Hz, H-
0
0
0
0
0
0
50a), 4.01 (dd, 1H, H-50b), 2.40 (s, 3H, PhCH3), 1.63, 1.33, 1.32, 1.29
(4s, 3H each, 4CH3); 13C NMR (125.7 MHz, CDCl3):
d 184.0 (C-2),
23 þ60 (c 1.21, CH2Cl2); 1H NMR (300 MHz, CDCl3):
167.9 (C-4), 139.6, 130.4, 129.9, 128.1 (Ar),114.7, 110.5 (2CMe2), 105.8
(C-8), 85.7 (C-9), 82.9 (C-6), 73.5 (C-40), 71.7 (C-5), 67.5 (C-50), 26.9,
26.6, 25.4 (4CH3), 21.4 (CH3Ar); HRLSI-MS m/z calcd for
C21H26N2NaO6S, [MþNa]þ: 457.1409, found: 457.1431.
(91 mg, 68%). [
a
]
D
d
9.58 (s, 1H, NH), 7.52e7.45 (m, 3H, Ar), 7.33e7.31 (m, 2H, Ar), 5.95
0
(d,1H, J8,9¼3.6 Hz, H-8), 4.77 (d,1H, H-9), 4.42 (d,1H, J6,4 ¼9.3 Hz, H-
6), 4.22 (ddd, 1H, J4 ,5 a¼5.7 Hz, J4 ,5 b¼3.6 Hz, H-40), 4.12 (dd, 1H,
0
0
0
0
J5 a,5 b¼9.0 Hz, H-50a), 4.02 (dd, 1H, H-50b), 1.64 (s, 3H, CH3), 1.33 (s,
0
0
4.2.3. (5S,6S,8R,9R,40R)-6-(20,20-Dimethyl-10,30-dioxolan-40-yl)-8,9-
(dimethylmethylenedioxy)-3-[200-(1000H-indol-3000-yl)ethyl]-2-thioxo-
7-oxa-1,3-diazaspiro[4.4]nonan-4-one (20). Method A: x¼25 ꢀC,
y¼24 h. Column chromatography: 99:1 CH2Cl2/MeOH. Amorphous
6H, 2CH3), 1.30 (s, 3H, CH3); 13C NMR (75.5 MHz, CDCl3):
d 183.7 (C-
2), 167.9 (C-4), 132.9, 129.4, 129.2, 128.3 (Ar), 114.6, 110.4 (2CMe2),
105.8 (C-8), 85.7 (C-9), 82.9 (C-6), 73.4 (C-40), 71.7 (C-5), 67.4 (C-50),
26.9, 26.5, 25.4 (4CH3); HRLSI-MS m/z calcd for C20H24N2NaO6S,
[MþNa]þ: 443.1253, found: 443.1227.
solid (107.8 mg, 79%). [
CDCl3):
a
]
23 þ70 (c 1.08, CH2Cl2); 1H NMR (300 MHz,
D
d
8.81 (s, 1H, NH), 8.10 (s, 1H, NH), 7.84 (d, 1H, JH,H¼7.5 Hz,
4.2. General procedures for the preparation of
thiohydantoins 18e24, 26e28
Ar), 7.36 (d, 1H, JH,H¼8.1 Hz, Ar), 7.25e7.11(m, 3H, Ar), 5.98 (d, 1H,
J8,9¼3.6 Hz, H-8), 4.55 (d, 1H, H-9), 4.42 (m, 1H, H-6), 4.17e3.99 (m,
5H, H-40, H-50a, H-50b, H-100), 3.13 (m, 2H, H-200), 1.67, 1.35, 1.34, 1.23
Method A: To
a
solution of isothiocyanate
4
(100 mg,
(4s, 3H each, 4CH3); 13C NMR (75.5 MHz, CDCl3):
d 183.5 (C-2), 168.4
0.28 mmol) in THF (1.5 mL) was added the corresponding amine
(0.42 mmol for thiohydantoins 18e22, 0.14 mmol for dimeric
thiohydantoins 26 and 27 and 0.046 mmol for trimeric
(C-4), 136.2, 127.4, 122.3, 122.2, 119.6, 119.1 (Ar), 114.6 (CMe2), 112.2,
111.2 (Ar) 110.3 (CMe2), 105.5 (C-8), 85.5 (C-9), 82.3 (C-6), 73.2 (C-
40), 71.1 (C-5), 67.3 (C-50), 42.2 (C-100), 26.7, 26.6, 26.4, 25.2 (4CH3),

9970
C. Gasch et al. / Tetrahedron 66 (2010) 9964e9973
23.1 (C-200); HRLSI-MS m/z calcd for C24H29N3NaO6S, [MþNa]þ:
added and the solution was heated at 60 ꢀC for 12 h. Column
chromatography: 4:1 Et2O/hexane. Amorphous solid (74 mg, 23%).
510.1675, found: 510.1684.
[
a
]
24 þ238 (c 0.48, CH2Cl2); 1H NMR (300 MHz, CDCl3) 6:1 atropi-
D
4.2.4. (5S,6S,8R,9R,40R)-3-Butyl-6-(20,20-dimethyl-10,30-dioxolan-40-
yl)-8,9-(dimethylmethylenedioxy)-2-thioxo-7-oxa-1,3-diazaspiro
[4.4]nonan-4-one (21). Method A: x¼25 ꢀC, y¼1 h. Column chro-
matography: 4:1/7:3 hexane/EtOAc gradient. Amorphous solid
someric mixture: major atropisomer:
d
00 00
6.47 (dd, 1H, J2 ,3 ¼12.0 Hz,
00 00
00
00
00 00
J3 ,4 ¼9.0 Hz, H-3 ), 6.38 (d, 1H, J1 ,2 ¼3.0 Hz, H-1 ), 5.98 (d, 1H,
00
00 00
J8,9¼4.0 Hz, H-8), 5.10 (dd, 1H, J4 ,5 ¼10.0 Hz, H-4 ), 5.04 (dd, 1H, H-
200), 4.53 (d, 1H, H-9), 4.39 (d, 1H, J6,4 ¼7.0 Hz, H-6), 4.32 (dd, 1H,
0
23 þ44 (c 1.34, CH2Cl2); 1H NMR (500 MHz, CDCl3):
J5 ,6 a¼4.0 Hz, J6 a,6 b¼11.0 Hz, H-600a), 4.20 (ddd, 1H, J4 ,5 ¼10.0 Hz,
00 00
00
00
00 00
(93 mg, 84%). [a]
D
J5 ,6 b¼2.0 Hz, H-500), 4.12 (m, 1H, H-40), 4.10 (d0d, 1H, H-600b), 4.06
00 00
d
9.05 (m, 1H, NH), 5.98 (d, 1H, J1,2¼3.5 Hz, H-8), 4.63 (d, 1H, H-9),
0
0
0
0
0
0
4.36 (d, 1H, J6,4 ¼9.0 Hz, H-6), 4.11 (ddd, 1H, J4 ,5a¼6.0 Hz,
(ddd, 1H, J5 a,4 ¼6.4 Hz, J5a ,5b ¼8.8 Hz, H-5 a), 3.85 (dd, 1H,
J4 ,5b¼3.5 Hz, H-40) 4.08 (dd, 1H, J5a ,5b ¼8.5 Hz, H-5 a), 3.98 (dd, 1H,
H-50b), 3.77 (m, 2H, H-100),1.68e1.59 (m, 2H, H-200),1.65 (s, 3H, CH3),
1.38 (m, 2H, H-30000), 1.32, 1.31, 1.22 (3s, 3H each, 3CH3), 0.94 (t, 3H,
J4 ,5 b¼5.5, H-50b), 2.11, 2.09, 2.02, 1.93 (4s, 3H each, 4COCH3), 1.58,
0
0
0
0
0
0
1.32, 1.28, 1.26 (4s, 3H each, 4CH3); minor atropisomer:
d 6.23 (d,
00
00 00
1H, J1 ,2 ¼3.2 Hz, H-1 ), 5.91 (d, 1H, J8,9¼3.3 Hz, H-8), 4.78 (dd, 1H,
J3 ,4 ¼7.5 Hz, H-4 ); 13C NMR (125.7 MHz, CDCl3):
d
183.9 (C-2),
J2 ,3 ¼11.8 Hz, H-2 ), 4.67 (d, 1H, H-9); 1H NMR (300 MHz, DMSO-
00
0
0
00 00
000,400
00 00
168.5 (C-4), 114.6, 110.4 (2CMe2), 105.7 (C-8), 85.9 (C-9), 82.3 (C-6),
73.3 (C-40), 71.1 (C-5), 67.4 (C-50), 41.6 (C-100), 29.5 (C-200), 26.9, 26.6,
26.5, 25.3 (4CH3), 20.0 (C-300), 13.8 (C-400); HRLSI-MS m/z calcd for
C18H28N2NaO6S, [MþNa]þ: 423.1566, found: 423.1583.
d6):
d
11.2 (br s, 1H, NH), 6.26 (dd, 1H, J2 ,3 ¼11.8 Hz, J3 ¼9.2 Hz,
H-300), 6.12 (d, 1H, J1 ,2 ¼3.0 Hz, H-1 ), 5.96 (d, 1H, J8,9 ¼3.8 Hz, H-8),
00
00 00
0
00
00
00 00
4.98 (t,1H, J4 ,5 ¼9.5 Hz, H-4 ), 4.91 (dd,1H, H-2 ), 4.60 (d,1H, H-9),
4.29e4.22 (m, 3H, H-5, H-500, H-6a00), 4.05e3.95 (m, 3H, H-40, H-6b00,
H-5a0), 3.84e3.80 (m, 1H, H-5b0), 2.09, 2.02, 1.95, 1.86 (4s, 3H each,
4OCH3), 1.42, 1.23, 1.20, 1.17 (4s, 3H each, 4CH3); 13C NMR
4.2.5. (5S,6S,8R,9R,40R)-3-Cyclohexyl-6-(20,20-dimethyl-10,30-dioxo-
lan-40-yl)-8,9-(dimethylmethylenedioxy)-2-thioxo-7-oxa-1,3-dia-
zaspiro[4.4]nonan-4-one (22). Method A: x¼25 ꢀC, y¼30 min.
(75.5 MHz, CDCl3) major atropisomer:
d 183.5 (C-2), 170.9, 170.1,
169.5, 169.0, 166.8 (C-4, 4C]O), 114.6, 110.3 (2CMe2), 104.8 (C-8),
89.9 (C-100), 88.0 (C-9), 80.5 (C-6), 73.0 (C-40), 71.0 (C-5), 70.1 (C-500),
69.8 (C-400), 66.8 (C-50), 66.4 (C-300), 61.8 (C-600), 57.0 (C-200), 27.3,
26.4, 26.2, 25.1 (4CH3), 21.5, 20.9, 20.8, 20.7 (4OCH3); minor atro-
Column chromatography: 80:1 CH2Cl2/MeOH. Amorphous solid
23
(112 mg, 94%). [
CDCl3):
a
]
D
þ54 (c 0.94, CH2Cl2); 1H NMR (500 MHz,
d
7.26 (s, 1H, NH), 5.96 (d, 1H, J8,9¼4.0 Hz, H-8), 4.62 (d, 1H,
H-9), 4. 44 (tt, 1H, JHH ¼12.0 Hz, JHH ¼4.0 Hz, CH cyclohexyl), 4.32
(m, 1H, H-6), 4.12e4.07 (m, 2H, H-40, H-50a), 3.96 (m, 1H, H-50b),
2.31 (dq, 1H, JHH¼12.5 Hz, JHH¼4.0 Hz, CHA), 2.16 (dq, 1H,
JHH¼12.2 Hz, JHH¼3.5 Hz, CHA), 1.83 (m, 2H, 2CHA) 1.73e1.67 (m, 2H,
2CHB), 1.70e1.64 (m, 1H, CHA), 1.64, 1.33, 1.32, 1.23 (4s, 3H each,
4CH3), 1.36e1.28 (m, 2H, 2CHB), 1.21e1.16 (m, 1H, CHB); 13C NMR
pisomer: d
104.9 (C-8), 91.2 (C-100), 86.3 (C-1), 70.3 (C-500), 66.9 (C-
0
0
400), 66.8 (C-50), 55.2 (C-200); HRLSI-MS m/z calcd for
C28H38N2NaO15S, [MþNa]þ: 697.1891, found: 697.1917.
4.2.8. (50S,60S,80R,90R,400R)-1,6-Bis[60-(200,200-dimethyl-100,300-dioxolan-
400-yl)-80,90-(dimethylmethylenedioxy)-40-oxo-20-thioxo-70-oxa-10,30-
diazaspiro[4.4]nonan-30-yl]hexane (26). Method A: x¼25 ꢀC, y¼2 h.
(127.5 MHz, CDCl3):
d 184.2 (C-2), 168.3 (C-4), 114.7, 110.4 (2CMe2),
105.6 (C-8), 85.8 (C-9), 82.5 (C-6), 73.3 (C-40), 70.0 (C-5), 67.4 (C-50),
55.8 (CH cyclohexyl), 28.8, 28.3 (2CH2), 26.9, 26.7, 26.6 (3CH3), 26.1,
26.0 (2CH2), 25.4 (CH3), 25.2 (CH2); HRLSI-MS m/z calcd for
C20H30N2NaO6S, [MþNa]þ: 449.1722, found: 449.1718.
Column chromatography: 40:1 CH2Cl2/MeOH. Amorphous solid
23
(101 mg, 93%). [
a
]
D
þ55 (c 0.72, CH2Cl2); 1H NMR (500 MHz,
0
0
0
CDCl3):
d
5.97 (d,1H, J8 ,9 ¼3.6 Hz, H-8 ), 5.29 (s,1H, NH), 4.6040 (d,1H,
H-90), 4.35 (d,1H, J6 ,4 ¼8.6 Hz, H-6 ), 4.09 (m, 2H, H-4 , H-5 a), 3.97
0
00
0
00
(m, 1H, H-500b), 3.78e3.71 (m, 2H, H-1), 1.68e1.64 (m, 2H, H-2), 1.65
4.2.6. (5S,6S,8R,9R,40R)-6-(20,20-Dimethyl-10,30-dioxolan-40-yl)-8,9-
(dimethylmethylenedioxy)-3-(200,300,400,600-tetra-O-acetyl-
b-D-gluco-
pyranosyl)-2-thioxo-7-oxa-1,3-diazaspiro[4.4]nonan-4-one
(23). Method B: x¼40 ꢀC, y¼12 h. Column chromatography: 3:2
(s, 3H, CH3),1.40 (m, 2H, H-3),1.33,1.31,1.23 (3s, 3H each, 3CH3); 13
C
NMR (125.7 MHz, CDCl3):
d
183.7 (C-20), 168.4 (C-40), 114.6, 110.4
(2CMe2), 105.6 (C-80), 85.8 (C-90), 82.3 (C-60), 73.3 (C-400), 71.1 (C-50),
67.4 (C-500), 41.6 (C-1), 27.3, 26.9, 26.6, 26.5, 26.4, 25.4 (C-2, C-3, 4
CH3); HRLSI-MS m/z calcd for C34H50N4NaO12S2, [MþNa]þ:
793.2754, found: 793.2746.
23
Et2O/hexane. Amorphous solid (142 mg, 76%). [
a
*
]
þ9 (c 1.17,
D
CH2Cl2); 1H NMR (500 MHz, CDCl3): roughly 1:1
atropisomeric
mixture:
d
6.43 (m, 1H, H-200), 6.04e6.95 (m, 3H, H-8, H-8
*
, H-100),
5.82 (m, 1H, H-200
*
)0,0 5.46 (m, 1H, H-100
*
), 5.29 (t, 00 2H,
00
4.2.9. (50S,60S,80R,90R,400R)-1,12-Bis[60-(200,200-dimethyl-100,300-dioxo-
lan-400-yl)-80,90-(dimethylmethylenedioxy)-40-oxo-20-thioxo-70-oxa-
10,30-diazaspiro[4.4]nonan-30-yl]dodecane (27). Method A: x¼25 ꢀC,
00 00
00 00
00 00
J2 ,3 ¼J3 ,4 ¼9.5 Hz, H-3 , H-3
*
), 5.22 (t, 2H, J4 ,5 ¼9.5 Hz, H-4 , H-
400
*
), 4.62 (m, 2H, H-9, H-9
*
), 4.37 (d, 2H, J6,4 ¼8.5 Hz, H-6, H-6
*
),
0
4.31 (br d, 2H, J6 a,6 b¼12.0 Hz, H-600a, H-600a
*
), 4.12 (dd, 2H,
00
00
J5 ,6 b¼5.0 Hz, H-600b, H-600b
*
), 4.14e4.11 (m, 4H, H-40, H-40 , H-50a,
*
y¼16 h, then, x¼40 ꢀC, y¼1 h. Column chromatography: 50:1
00 00
H-50a
*
), 4.01 (m, 2H, H-50b, H-50b
*
), 3.81 (ddd, 2H, J5 ,6 a¼2.5 Hz, H-
24
00 00
CH2Cl2/MeOH. Amorphous solid (105 mg, 88%). [
a]
þ35 (c 1.27,
D
500, H-500
6H, CH3, CH3
CH3
); 13C NMR (125.7 MHz, CDCl3):
(ꢁ2), 170.2, 170.0 (ꢁ3), 169.5, 169.2, 168.3, 165.9 (C-4, C-4
4C]O ), 114.9, 114.3, 111.2, 110.4 (2CMe2, 2CMe2), 106.3, 104.7 (C-8,
), 82.5 (C-6), 81.6 (C-100), 79.7 (C-100
C-8 ), 86.6, 86.2 (C-9, C-9 ), 79.5
(C-6
), 74.3 (C-500), 73.5 (C-500
70.4 (C-5, C-5
), 68.6, 68.4 (C-200, C-200
(C-50, C-50 ), 62.0 (C-600, C-600
(4CH3, 4CH3); 20.9, 20.7 (4COCH3, 4COCH3); HRLSI-MS m/z calcd for
*
), 2.06, 2.05, 2.00, 1.95 (4s, 6H each, 4AcO, 4AcO
), 1.35e1.29 (m, 12H, 2CH3, 2CH3), 1.25 (s, 6H, CH3,
182.7, 180.6 (C-2, C-2 ), 170.7
, 4C]O,
*
), 1.62 (s,
CH2Cl2); 1H NMR (500 MHz, CDCl3): d08.68 (s, 1H, NH), 5.97 (d, 1H,
0
0
*
*
0
0
J8 ,9 ¼4.0 Hz, H-8 ), 4.63 (d, 1H, H-9 ), 4.38e4.35 (m, 1H, H-6 ),
4.12e4.07 (m, 2H, H-400, H-500a), 4.00e3.96 (m, 1H, H-500b), 3.76 (m,
2H, H-1), 1.68e1.60 (m, 2H, H-2), 1.65 (s, 3H, CH3), 1.36e1.22 (m, 8H,
H-3eH-6), 1.33, 1.31, 1.23 (3s, 3H each, 3CH3); 13C NMR (125.7 MHz,
*
d
*
*
*
*
CDCl3): d
183.9 (C-20), 168.4 (C-40), 114.7, 110.4 (2CMe2), 105.6 (C-80),
*
*
*
), 72.8 (C-300, C-300 , C-40, C-40 ), 71.6,
*
85.9 (C-90), 82.3 (C-60), 73.4 (C-400), 71.1 (C-50), 67.4 (C-500), 41.9 (C-
1), 29.7, 29.6, 29.3 (3CH2), 27.4, 26.9, 26.8, 26.6, 26.5 (2CH2, 3CH3),
25.3 (CH3); HRLSI-MS m/z calcd for C40H62N4NaO12S2, [MþNa]þ:
877.3703, found: 877.3712.
*
*
*
*
), 68.1, 67.5 (C-400, C-400*
), 66.4
*
*
*
), 26.9 (ꢁ2), 26.6 (ꢁ3), 26.1, 25.1, 25.0
*
*
C28H38N2NaO15S, [MþNa]þ: 697.1891, found: 697.1893.
4.2.10. (50S,60S,80R,90R,400R)-Tris(2-[60-(200,200-dimethyl-100,300-dioxolan-
400-yl)-80,90-(dimethylmethylenedioxy)-40-oxo-20-thioxo-70-oxa-10,30-di-
azaspiro[4.4]nonan-30-yl]ethyl)amine (28). Method A: x¼25 ꢀC,
y¼24 h. Column chromatography: 20:1 CH2Cl2/MeOH. Amorphous
4.2.7. (5S,6S,8R,9R,40R)-6-(20,20-dimethyl-10,30-dioxolan-40-yl)-8,9-
(dimethylmethylenedioxy)- 3-(100,300,400,600-tetra-O-acetyl-200-deoxy-
b-
d-glucopyranos-200-yl)-2-thioxo-7-oxa-1,3-diazaspiro[4.4]nonan-4-one
(24). Method B: x¼40 ꢀC, y¼12 h. Then, more Et3N (0.27 mmol) was

C. Gasch et al. / Tetrahedron 66 (2010) 9964e9973
9971
23
00
0
00 00
0
solid (51 mg, 97%). [
a
]
þ50 (c 1.03, CH2Cl2); 1H NMR (300 MHz,
J2 ,3 ¼8.5 Hz, H-2 ), 4.30 (d,1H, J6,4 ¼8.8, H-6), 4.13 (m,1H, H-4 ), 4.10
D
0
(dd,1H, J4 ,5 a¼5.7 Hz, J5a ,5 b¼11.4 Hz, H-50a), 4.02 (m,1H, H-50b), 3.88
0
0
0
0
0
0
CDCl3):
d
8.62 (m,1H, NH), 5.98 (d,1H, J8 e9 ¼3.6 Hz, H-8 ), 4.70 (d,010 H,
H-90), 4.35 (d, 1H, J6 ,4 ¼8.1 Hz, H-6 ), 4.11e4.07 (m, 2H, H-4 , H-5 a),
4.00e3.84 (m, 3H, H-500b, CH2), 2.86 (m, 2H, CH2), 1.63 (s, 3H, CH3),
1.33 (s, 6H, 2CH3), 1.25 (s, 3H, CH3); 13C NMR (75.5 MHz, CDCl3):
(d,1H, J5 ,6 a¼12.3 Hz, H-600a), 3.69 (m,1H, H-600b), 3.46e3.41 (m, 3H,
0
00
0
00
00 00
H-300, H-400, H-500), 1.58, 1.44, 1.33, 1.28 (4s, 3H each, 4CH3); 13C NMR
(75.5 MHz, CD3OD):
d 184.5 (C-2), 169.9 (C-4), 115.4, 112.1 (2CCH3);
d
183.3 (C-20), 168.2 (C-40), 114.6, 110.4 (2CMe2), 105.6 (C-80), 85.8 (C-
107.0 (C-8), 86.8 (C-9), 85.5 (C-100), 83.3 (C-6), 81.2, 78.7 (C-300, C-500),
74.3 (C-40), 71.2 (C-400), 70.9 (C-5), 69.9 (C-200), 67.6 (C-50), 63.3 (C-600),
26.9 (ꢁ2), 26.7, 25.2 (4CH3); HRCI-MS m/z calcd for C20H31N2O11S,
[MþH]þ: 507.1649, found: 507.1641.
90), 82.2 (C-60), 73.3 (C-400), 71.1 (C-50), 67.3 (C-500), 50.6 (C-1) 39.7 (C-
2), 26.9, 26.6, 26.5, 25.2 (4CH3); HRLSI-MS m/z calcd for
C48H69N7NaO18S3, [MþNa]þ: 1150.3759, found: 1150.3719.
4.2.11. N-(2,3,4-Tri-O-acetyl-
1,2:5,6-di-O-isopropylidene-3-methoxycarbonyl-
a
-
D
-arabinopyranosyl)-N0-(3S-3-deoxy-
-ribo-hexafur-
4.3. General procedure for the deprotection of thiohydantoins
a-D
anos-3-yl)thiourea (13). To a solution of aminoester 3 (61 mg,
0.19 mmol) in THF (2 mL) was added commercially available 2,3,4-
A solution of the protected thiohydantoins (x mg) in 9:1 TFAeH2O
(y mL) was kept stirring at rt for z h. Next, the solvent was removed
under reduced pressure co-evaporating with toluene several times
tri-O-acetyl-
a
-
D
-arabinopyranosyl
isothiocyanate
(66
mg,
0.21 mmol,1.10 equiv) and the reaction was kept stirring at 40 ꢀC for
3.5 h and at 60 ꢀC for 6 h. The solvent was removed under reduced
pressure and the residue was purified by column chromatography
(9:1 hexane/Et2O/Et2O) to afford 13 as an amorphous solid
to give the unprotected thiohydantoins as a/b mixtures.
4.3.1. (5S,6R,9R,10S)-6,7,10-Trihydroxy-9-hydroxymethyl-3-phenyl-
2-thioxo-8-oxa-1,3-diazaspiro[4.5]decan-4-one mg
(30). x¼47
23
(160.2 mg, 81%). [
a
]
D
23 0 (c 1.09, CH2Cl2); 1H NMR (300 MHz, CDCl3):
(0.11 mmol); y¼5 mL; z¼5 h. Amorphous solid (35 mg, 92%). [
a]
D
d
7.97 (d, 1H, JNH,1¼6.6, NH), 7.31 (s, 1H, NH’), 6.03 (d, 1H,
þ11 (c 1.01, CH3OH). 1H NMR (300 MHz, CD3OD)
b anomer:
0
0
0
J1 ,2 ¼3.9 Hz, H-1 ), 5.64 (t, 1H, J1,2¼9.6 Hz, H-1); 5.33 (m, 1H, H-4),
5.20 (t, 1H, J2,3¼9.6 Hz, H-2), 5.12 (dd, 01H, J3,4¼3.3 Hz, H-3), 4.82 (d,
d 7.45e7.36 (m, 3H, Ar), 7.25e7.22 (m, 2H, Ar), 5.30 (d, 1H,
0
0
J6,7¼8.1 Hz, H-7), 4.17 (dd,1H, J9,10¼10.2 Hz, J9,1 a¼0 Hz, J9,1 b¼4.8 Hz,
1H, H-20), 4.60 (d,1H, J4 ,5 ¼8.1 Hz, H-40), 4.23 (m,1H, H-5 ), 4.12 (dd,
H-9), 3.83 (d, 1H, J1a ,1 b¼11.7 Hz, H-10a), 3.75 (d, 1H, H-10), 3.64 (dd,
0
0
0
0
0
1H, J5 ,6a ¼6.3 Hz, J6a ,6b ¼9.0 Hz, H-6a ), 4.01 (dd, 1H, J5 ,6b ¼4.5 Hz,
H-6b0), 3.96 (dd, 1H, J4,5a¼1.5 Hz, J5a-5b¼12.9 Hz, H-5a); 3.81 (s, 3H,
OMe), 3.76 (d, 1H, H-5b); 2.10 (ꢁ2), 2.0 (3s, 3H each, 3AcO), 1.53,
1.51, 1.44, 1.29 (3s, 3H each, 3CH3); 13C NMR (75.5 MHz, CDCl3):
1H, H-10b), 3.47 (d, 1H, H-6);
a
anomer:
d
5.09 (d, 1H, J6,7¼4.7, H-7),
0
0
0
0
0
0
3.88 (d,1H, H-6); 13C NMR (75.5 MHz, CD3OD)
2), 174.5 (C-4), 135.0, 129.8, 129.7, 129.6 (Ar), 94.9 (C-7), 74.8, 74.7
b anomer: d 185.8 (C-
(C-9, C-5), 74.4 (C-6), 70.3 (C-10), 62.6 (C-10);
a anomer: d 94.6 (C-
d
184.8 (C]S), 170.7, 170.2, 169.9, 166.2 (4C]O), 113.3, 110.9
7), 70.1 (C-6); HRCI-MS m/z calcd for C14H17N2O6S, [MþH]þ:
(2ꢁCMe2), 104.1 (C-10), 88.4 (C-20), 84.6 (C-1), 80.8 (C-40), 72.4, 72.3
(C-30, C-50), 71.3 (C-3), 68.5 (C-4), 67.8 (C-2), 67.2 (C-60), 66.2 (C-5),
53.0 (OMe), 26.8, 26.2 (ꢁ2), 25.2 (4ꢁCH3), 21.0, 20.7 (3COMe);
HRLSI-MS m/z calcd for C26H38N2NaO14S, [MþNa]þ: 657.1941,
found: 657.1934.
341.0807, found: 341.0800.
4.3.2. (5S,6R,9R,10S)-3-Butyl-6,7,10-trihydroxy-9-hydroxymethyl-2-
thioxo-8-oxa-1,3-diazaspiro[4.5]decan-4-one
(31). x¼141
mg
(0.35 mmol); y¼15 mL; z¼6 h. Amorphous solid (142 mg, 88%).
23
[a
]
þ25 (c 0.92, CH3OH); 1H NMR (300 MHz, DMSO-d6):
D
4.2.12. (5S,6S,8R,9R,40R)-6-(20,20-Dimethyl-10,30-dioxolan-40-yl)-8,9-
b anomer: 10.10 (s, 1H, NH), 7.30e5.70 (m, 4H, 4OH), 5.04 (d, 1H,
(dimethylmethylenedioxy)-2-thioxo-3-(200,300,400-tri-O-acetyl-
b
-d-
J6,7¼7.8 Hz, H-7), 3.90 (ddd, 1H, J9,10¼9.9 Hz, J9,1 a¼2.0, J9,1 b¼5.4 Hz,
0
0
arabinopyranosyl)-7-oxa-1,3-diazaspiro[4.4]nonan-4-one
(25). A
H-9) 3.66e3.63 (m, 1H, H-10a), 3.60e3.56 (m, 2H, H-100), 3.47 (d, 1H,
H-10), 3.37 (dd, 1H, J1 a,1 b¼12.0 Hz, H-10b), 3.24 (d, 1H, H-6),
1.56e1.44 (m, 2H, H-200), 1.35e1.19 (m, 2H, H-300), 0.85 (t, 3H,
0
0
solution of tiourea 13 (160.2 mg, 0.25 mmol) in THF (2 mL) was
refluxed for 2 h; then the solvent was removed under reduced
pressure and the residue was purified by column chromatography
(60:1 CH2Cl2/MeOH) to give 25 as an amorphous solid (141.5 mg,
JH,H¼7.3 Hz, CH3);
J6,7¼3.2 Hz, H-7), 3.91e3.85 (m, 1H, H-9), 0.86 (t, 3H, JH,H¼7.3 Hz,
CH3); 13C NMR (75.5 MHz, DMSO-d6):
anomer: 182.9 (C-2), 173.3
(C-4), 93.4 (C-7), 73.5 (C-9), 72.5 (C-6), 72.2 (C-5), 68.2 (C-10), 60.8
(C-10), 40.3e38.6 (C-100), 29.4, 19.1 (2CH2), 13.7 (CH3);
anomer:
a anomer: d 10.36 (s, 1H, NH), 4.90 (d, 1H,
93%). [
a
]
23 þ30 (c 1.11, CH2Cl2); 1H NMR (300 MHz, CDCl3):
d
8.21 (s,
b
d
D
00
00 00
00 00
1H, NH), 6.14 (br t, 1H, J1 ,2 ¼J2 ,3 ¼9, H-2 ), 5.93 (d, 1H, J8,9¼3.6 Hz,
H-8), 5.68 (d, 1H, H-100), 5.25 (m, 1H, H-400), 5.16 (dd, 1H,
a
00
00
00
0
J3 e4 ¼3.3 Hz, H-3 ), 4.61 (d, 1H, H-9), 4.41 (d, 1H, J6,4 ¼8.4 Hz, H-6),
d 182.5 (C-2), 176.4 (C-4), 92.8 (C-7), 74.1 (C-5), 68.0 (C-6), 67.9 (C-
4.10e4.01 (m, 3H, H-40, H-50a, H-500a), 3.98e3.94 (m, 1H, H-50b),
9), 67.3 (C-10), 60.3 (C-10), 29.3 (CH2); HRCI-MS m/z calcd for
C12H21N2O6S, [MþH]þ: 321.1120, found: 321.1110.
3.73 (d, 1H, J5 a,5 b¼12.9, H-500b), 2.17, 2.00,1.99 (3s, 3H each, 3AcO);
00
00
1.67,1.38,1.30,1.21 (4s, 3H each, 4CH3); 13C NMR (75.5 MHz, CDCl3):
d
182.7 (C-2); 170.9, 170.2 (ꢁ2), 166.4 (C-4, 3C]O); 114.9, 110.6
4.3.3. (5S,6R,9R,10S)-3-Cyclohexyl-6,7,10-trihydroxy-9-hydrox-
ymethyl-2-thioxo-8-oxa-1,3-diazaspiro[4.5]decan-4-one
(2CMe2),105.5 (C-8), 85.6 (C-9), 82.4 (C-6, C-100), 73.4 (C-40), 71.3 (C-
300), 70.0 (C-5), 68.1 (C-400), 67.3 (C-50), 66.7 (C-200), 66.1 (C-500), 27.0,
26.8, 25.7, 25.1 (4CH3); 21.1, 20.8 (ꢁ2) (3COCH3); HRLSI-MS m/z
calcd for C25H34N2NaO13S, [MþNa]þ: 625.1679, found: 625.1695.
(32). x¼140 mg (6.33 mmol); y¼15 mL; z¼3h. Amorphous solid
23
(112.8 mg, 99%). [
DMSO-d6):
a
]
þ22 (c 0.75, CH3OH); 1H NMR (500 MHz,
D
b anomer: d 10.05 (s, 1H, NH), 6.74 (br s, 1H, OH-7),
5.64e5.60 (m, 2H, OH-6, OH-10), 5.04 (d, 1H, J6,7¼8.0 Hz, H-7), 4.49
4.2.13. (5S,6S,8R,9R,40R)-6-(20,20-Dimethyl-10,30-dioxolan-40-yl)-8,9-
(br s, 1H, OH-10), 4.45e4.36 (m, 1H, H-100), 3.90 (ddd, 1H,
0
0
(dimethylmethylenedioxy)-3-
b
-
D
-glucopyranosyl-2-thioxo-7-oxa-
J9,10¼9.5 Hz, J9,1 a¼2.0 Hz, J9,1 b¼5 Hz, H-9), 3.61 (dd, 1H,
J1 a,1 b¼12.0 Hz, H-10a), 3.45 (dd, 1H, J10,OH¼5.0 Hz, H-10), 3.38 (dd,
1H- H-10b), 3.22 (d, 1H, H-6), 2.14e2.09 (m, 2H, CH2), 1.76e1.74 (m,
2H, CH2), 1.61e1.47 (m, 4H, 2CH2), 1.25e1.17 (m, 2H, CH2);
0
0
1,3-diazaspiro[4.4]nonan-4-one (29). To a solution of thiohydantoin
23 (71 mg, 0.11 mmol) in anhydrous MeOH (4 mL) was added NaOMe
(24 mg, 0.44 mmol) and the corresponding mixture was stirred at rt
for 1.5 h. Then it was neutralized with Amberlite IR-120(Hþ) resin
and filtrated. The filtrate was concentrated to dryness and the resi-
due was purified by column chromatography (40:1/10:1 CH2Cl2/
a
anomer:
d
10.33 (s, 1H, NH), 6.24 (d, 1H, J7,OH¼11.0 Hz, OH-7), 5.90
(d, 1H, J10,OH¼6.0 Hz, OH-10), 5.76 (br s, 1H, OH-6), 4.88 (dd, 1H,
J6,7¼4.0 Hz, H-7), 4.45e4.36 (m, 1H, OH-10), 3.86 (ddd, 1H,
23 þ56 (c
J9,10¼10.1 Hz, J9,1 a¼2.0 Hz, J9,1 b¼5.5 Hz, H-9), 3.65e3.62 (m, 1H, H-
0
0
MeOH) to give 29 as an amorphous solid (29.5 mg, 56%). [
a
]
D
0.85, MeOH); 1H NMR (300 MHz, CD3OD):
d
5.98 (d, 1H, J8,9¼3.6 Hz,
6), 3.59 (dd,1H, J1 a,1 b¼12.0 Hz, H-10a), 3.55 (dd,1H, H-10), 3.48 (dd,
0
0
1H- H-10b); 13C NMR (125.7 MHz, DMSO-d6):
b anomer: d 183.3
00
00 00
H-8), 5.69 (d,1H, J1 ,2 ¼9.3 Hz, H-1 ), 4.68 (d,1H, H-9), 4.38 (br t,1H,

9972
C. Gasch et al. / Tetrahedron 66 (2010) 9964e9973
(C-2), 173.4 (C-4), 93.3 (C-7), 73.4 (C-9), 72.4 (C-6), 71.3 (C-5), 68.4
(C-10), 61.0 (C-10), 53.6 (C-100), 28.3, 25.5, 24.9 (4CH2);
anomer:
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, respectively. 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/Kmꢂ1), were [I] is the
a
d
182.9 (C-2), 176.7 (C-4), 92.8 (C-7), 73.1 (C-5), 68.0 (C-6), 67.7 (C-
9), 67.5 (C-10), 60.4 (C-10), 54.0 (C-100), 28.1, 25.4, 24.8 (3CH2); HRCI-
MS m/z calcd for C14H23N2O6S, [MþH]þ: 347.1277, found: 347.1284.
4.3.4. (50S,60R,90R,100S)-1,6-Bis-(60,70,100-trihydroxy-90-hydrox-
ymethyl-40-oxo-20-thioxo-80-oxa-10,30-diazaspiro[4.5]decan-30-yl)
0
hexane (33). x¼60 mg (0.08 mmol); y¼6.4 mL; z¼5 h. Amorphous
inhibitor concentration in the cuvette and Km and Km are the enzy-
23
solid (48 mg, 99%). [
a
]
D
þ15 (c 1.08, CH3OH); 1H 0NMR (500 MHz,
matic MichaeliseMenten constants in the absence and in the pres-
ence of the inhibitor, respectively.
0
0
CD3OD)
b
anomer:
d
00
5.36 (d, 1H, J6 ,7 ¼8.1 Hz, H-7 ), 4.23 (ddd, 1H,
J9 ,10 ¼10.2 Hz, J9 ,1 a¼2.1 Hz, J9 ,1 b¼5.1 Hz, H-90), 3.88 (dd, 1H,
Glycogen phosphorylase activities were assayed in the direction
0
0
0
0
00
J1 a,1 b¼12.6 Hz, H-100a), 3.83 (d, 1H, H-100), 3.77 (t, 2H, J1,2¼6.9 Hz,
of glycogen synthesis34 by using commercial glycogen phosphor-
00
00
H-1), 3.68 (dd, 1H, H-100b), 3.56 (d, 1H, H-60), 1.61 (quint, 2H,
ylase b (rabbit muscle, 10
oyster glycogen, -glucose-1-phosphate, 1 mM AMP, with or
m
g/mL) at 30 ꢀC in the presence of 1%
J2,3¼6.9 Hz, H-2), 1.33 (m, 2H, H-3);
a
anomer:
d
5.22 (d, 1H,
a-D
J6 ,7 ¼4.5 Hz, H-7 ), 4.26 (m, 1H, H-9 ) 4.02 (d, 1H, H-6 ); 13C NMR
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.35
Hanes plots were built using the initial velocity from each re-
action, as indicated above for glycosidases.
0
0
0
0
0
(75.5 MHz, D2O)
b anomer: d
185.6 (C-20), 174.9 (C-40), 94.9 (C-70),
73.7, 73.6 (C-90, C-50), 72.7 (C-60), 68.7 (C-100), 62.2 (C-100), 41.4 (C-
1), 27.3, 25.8 (C-2, C-3);
a
anomer: 93.1 (C-70), 74.8 (C-50), 68.0 (C-
90), 67.8 (C-10), 60.8 (C-100); HRLSI-MS m/z calcd for
C22H34N4O12S2Na, [MþNa]þ: 633.1520, found: 633.1512.
Acknowledgements
4.3.5. (50S,60R,90R,100S)-1,12-Bis-(60,70,100-trihydroxy-90-hydrox-
ymethyl-40-oxo-20-thioxo-80-oxa-10,30-diazaspiro[4.5]decan-30-yl)do-
ꢀ
ꢀ
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
decane (34). x¼108 mg (0.13 mmol); y¼11.5 mL; z¼5 h. Amorphous
23
solid (86 mg, 98%). [
a
]
þ7 (c 1.30, CH3OH); 1H NMR (500 MHz,
D
0
0
0
CD3OD)
b
anomer:
d
00
5.30 (d, 1H, J6 ,7 ¼8.0 Hz, H-7 ), 4.17 (ddd, 1H,
ꢀ
Fundacion Carolina of Spain for the award of a fellowship. We also
J9 ,10 ¼10.0 Hz, J9 ,1 a¼2.3 Hz, J9 ,1 b¼5.0 Hz, H-90), 3.82 (dd, 1H,
0
0
0
0
00
thank Professors P. Gergely and T. Docsa from the University of
Debrecen (Hungary) for providing valuable information concerning
glycogen phosphorylase assays.
J1 a,1 b¼12.0 Hz, H-100a), 3.73 (t, 2H, J1,2¼7.2 Hz, H-1), 3.51 (d, 1H, H-
00
00
100), 3.62 (dd, 1H, H-100b), 3.37 (d, 1H, H-60), 1.64e1.61 (m, 2H, CH2),
0
0
0
1.32e1.28 (m, 8H, 4CH2);
a
anomer:
d
5.05 (d,1H, J6 ,7 ¼4.5 Hz, H-7 ),
4.13 (ddd, 1H, J9 ,10 ¼10.0 Hz, J9 ,1 a¼3.0 Hz, J9 ,1 b¼4.5 Hz, H-90); 13C
0
0
0
00
0
00
Supplementary data
NMR (125.7 MHz, CD3OD)
b anomer: d
186.2 (C-20), 175.0 (C-40),
94.9 (C-70), 74.8 (C-90), 74.3 (C-50), 74.1 (C-60), 70.1 (C-100), 62.7 (C-
100), 41.7 (C-1), 30.7, 30.6, 30.4, 28.7, 27.6 (C-2eC-6);
anomer:
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.tet.2010.09.109.
a
d
94.8 (C-7), 75.7 (C-50), 69.3 (C-9), 64.9 (C-100); HRLSI-MS m/z calcd
for C28H46N4NaO12S2, [MþNa]þ: 717.2451, found: 717.2464.
References and notes
4.3.6. (50S,60R,90R,100S)-Tris[2-(60,70,100-trihydroxy-90-hydrox-
ymethyl-40-oxo-20-thioxo-80-oxa-10,30-diazaspiro[4.5]decan-30-yl)
ethyl]ammonium trifluoroacetate(35). x¼102 mg (0.09 mmol);
1. Romeo, G.; Chiacchio, U.; Corsaro, A.; Merino, P. Chem. Rev. 2010, 110,
3337e3370.
2. Adema, A. D.; Bijnsdorp, I. V.; Sandvold, M. L.; Verheul, H. M.; Peters, G. J. Curr.
Med. Chem. 2009, 16, 4632e4643.
3. Guo, H.-M.; Wu, Y.-Y.; Niu, H.-Y.; Wang, D.-C.; Qu, G.-R. J. Org. Chem. 2010, 75,
3863e3866.
4. Rodríguez, J. B.; Comin, M. J. Mini-Rev. Med. Chem. 2003, 3, 95e114.
y¼11.5 mL; z¼5 h. Amorphous solid (78 mg, 98%). [
a
]
D
23 þ14 (c 1.00,
CH3OH); 1H NMR (300 MHz, D2O)
b
anomer:
d
5.34 (d, 1H,
0
0
0
J6 ,7 ¼8.1 Hz, H-7 ), 4.29 (t, 2H, J1,2¼6 Hz, H-1), 4.21 (dd, 1H,
ꢀ
~
5. Fernandez-Bolanos, J. G.; Al-Masoudi, N. A. L.; Maya, I. Adv. Carbohydr. Chem.
Biochem. 2001, 57, 21e98.
J9 ,10 ¼9.9 Hz, J9 -1 a¼0.0 Hz, J9 -1 b¼5.1 Hz, H-90), 3.87 (d, 1H,
0
0
0
00
0
00
J1 a,1 b¼12.6 Hz, H-100a), 3.84 (d, 1H, H-100), 3.68 (dd, 1H, H-100b),
00
00
ꢀ
ꢀ
6. Boto, A.; Hernandez, D.; Hernandez, R. Eur. J. Org. Chem. 2010, 3847e3857.
7. Jeong, L. S.; Choi, Y. N.; Tosh, D. K.; Choi, W. J.; Kim, H. O.; Choi, J. Bioorg. Med.
Chem. 2008, 16, 9891e9897.
3.73e3.69 (m, 2H, H-2), 3.57 (d, 1H, H-60);
a anomer: d 5.24 (d, 1H,
J6 ,7 ¼4.8 Hz, H-7 ), 4.00 (d, 1H, H-6 ); 13C NMR (75.5 MHz, D2O)
0
0
0
0
8. (a) Chen, H.; Zhang, H.; Feng, J.; Li, X.; Jiao, L.; Qin, Z.; Yin, Q.; Zhang, J. Eur. J. Org.
Chem. 2009, 6100e6103; (b) Fuentes, J.; Angulo, M.; Pradera, M. A. J. Org. Chem.
2
b
anomer:
d
183.2 (C-20), 173.6 (C-4ꢂ0), 163.5 (q, JC,F¼35.6 Hz,
CF3COOꢂ), 116.9 (JC,F¼291.6 Hz, CF3COO ), 92.9 (C-70), 73.6, 73.0 (C-
50, C-90), 72.1 (C-60), 68.1 (C-100), 60.5 (C-100), 51.2 (C-2), 35.4 (C-1);
HRLSI-MS m/z calcd for C30H45N7NaO18S3 [MꢂTFAþNa]þ: 910.1881,
found: 910.1838.
2002, 67, 2577e2587; (c) Fernandez-Bolanos, J. G.; Zafra, E.; Lopez, O; Robina,
I.; Fuentes, J. Tetrahedron: Asymmetry 1999, 10, 3011e3023.
ꢀ
ꢀ
~
ꢀ
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