6-Azido hyacinthacine A2 gives a straightforward access to the first multivalent pyrrolizidine architectures

Article abstract of DOI:10.1039/c4ob01117a

The synthesis of the first multivalent pyrrolizidine iminosugars is reported. The key azido intermediates 4 and 31 were prepared after suitable synthetic elaboration of the cycloadduct obtained from 1,3-dipolar cycloaddition of d-arabinose derived nitrone

Full text of DOI:10.1039/c4ob01117a

Organic &
Biomolecular Chemistry
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6-Azido hyacinthacine A2 gives a straightforward
access to the first multivalent pyrrolizidine
architectures†
Cite this: Org. Biomol. Chem., 2014,
12, 6250
Giampiero D’Adamio,a,b Camilla Parmeggiani,a,b,c Andrea Goti,*a
Antonio J. Moreno-Vargas,d Elena Moreno-Clavijo,d Inmaculada Robinad and
Francesca Cardona*a
The synthesis of the first multivalent pyrrolizidine iminosugars is reported. The key azido intermediates 4
and 31 were prepared after suitable synthetic elaboration of the cycloadduct obtained from 1,3-dipolar
cycloaddition of D-arabinose derived nitrone to dimethylacrylamide. The key step of the strategy was the
stereoselective installation of an azido moiety at C-6 of the pyrrolizidine skeleton. The click reaction with
different monovalent and dendrimeric alkyne scaffolds allowed the preparation of a library of new mono-
and multivalent pyrrolizidine compounds that were preliminarily assayed as glycosidase inhibitors towards
a panel of commercially available glycosyl hydrolases.
Received 30th May 2014,
Accepted 18th June 2014
DOI: 10.1039/c4ob01117a
www.rsc.org/obc
effect on a glycosidase was recently reported for a trivalent
1-nojirimycin derivative that showed a 6-fold affinity enhance-
Introduction
The multivalent display of carbohydrate ligands on a scaffold ment towards jack bean α-mannosidase.5 Much greater effects
is currently an area of great interest, since carbohydrates bind were demonstrated for fullerene decorated with up to 12 deoxy-
only weakly to their complementary proteins, while modu- nojirimycin iminosugars6 and for cyclodextrin-based scaffolds
lation and enhancement of the biological response can be decorated with 7 and 14 deoxynojirimycin units.7 In both
achieved by means of multiple interactions established by cases, the highest inhibition enhancements were found with
multivalent carbohydrates. The multivalent or cluster glycoside jack bean α-mannosidase. Remarkably high multivalent effects
effect is defined as the affinity enhancement obtained with were determined recently with relatively low valent (4 epitopes)
multivalent ligands compared to their monovalent counter- porphyrin-based 1-deoxynojirimycin (1-DNJ) inhibitors,
parts.1 This concept, widely investigated for studying showing the importance of the spatial presentation of the DNJ
carbohydrate–lectin interactions,2 has remained essentially epitopes in modulating the activity (that is, the compounds
unexplored concerning specific glycosidase inhibition using with highest valencies are not necessarily the best inhibitors).8
iminosugars3 as glycomimetics up to 2010.
The multivalent approach towards α-L-fucosidase inhibition
Some examples of relatively low valent (2–4 iminosugar was recently explored by some of us with trivalent pyrrolidine
units) monocyclic iminosugars were already reported in the derivatives.9,10 Two mechanisms have been proposed for the
literature, but early glycosidase inhibition tests were not rationalization of the multivalent effect:11 the “statistical
encouraging.4 The first promising example of a multivalent rebinding” or “proximity effect”12 and the standard “chelation
effect”. The former appears more appropriate for glycosidases
since these types of enzymes have usually a single binding site
and therefore an increased propensity for ligand rebinding
may occur when additional ligands are in close proximity.
aDipartimento di Chimica “Ugo Schiff”, Università di Firenze, Via della Lastruccia
3-13, 50019 Sesto Fiorentino (FI), Italy. E-mail: francesca.cardona@unifi.it,
andrea.goti@unifi.it
bEuropean Laboratory for Non Linear Spectroscopy (LENS), via Nello Carrara 1,
However, very recent studies highlighted the existence of inter-
actions of the multivalent inhibitors with non-glycone sites,13
50019 Sesto Fiorentino (FI), Italy
cCNR-INO, U.O.S. Sesto Fiorentino, via Nello Carrara 1, 50019 Sesto Fiorentino (FI),
and the formation of aggregates of receptors with different
sizes and shapes.8
A strong multivalent effect was also observed with deoxyno-
jirimycin-decorated cyclodextrins towards a β-glucosidase of
therapeutic interest, namely human GCase, thus showing the
applicability of the multivalence concept in the development
Italy
dDepartamento de Química Orgánica, Facultad de Química, Universidad de Sevilla,
c/Prof. García González 1, E-41012 Sevilla, Spain
†Electronic supplementary information (ESI) available: 1H NMR and 13C NMR
spectra of all new compounds, IC50 and Lineweaver–Burk plots of active com-
pounds towards amyloglucosidase. See DOI: 10.1039/c4ob01117a
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of chaperones for the treatment of Gaucher’s disease.14 Con- evaluation towards a panel of commercial glycosidases is also
versely, in this regard it was recently shown that the com- reported herein.
pounds displaying the best affinity for GCase are not
necessarily the best chaperones, the best results in increasing
the enzyme activity being observed with relatively low valent
(tri- and tetravalent) deoxynojirimycin analogues. This could
be reasonably explained by a lower cellular permeability of
Results and discussion
Cycloaddition of D-arabinose derived nitrone 1 24 to N,N-
dimethylacrylamide afforded isoxazolidine 2 24a,18d with
cyclodextrin-based compounds with respect to smaller dendri-
meric analogues.15 Relatively small trivalent and tetravalent
improved 83% yield. This compound, after N–O bond cleavage
with Zn in acetic acid and reduction of the CvO bond with
N-butyl deoxynojirimycin analogues were also recently found
to act as more potent CFTR (cystic fibrosis transmembrane
conductance regulator) correctors than the corresponding
LiAlH4 in THF afforded compound 3 18d with an optimized
95% overall yield. Straightforward introduction of the azido
group was achieved by the direct Mitsunobu reaction of 3 with
monovalent iminosugars, thus representing promising new
candidates for the treatment of cystic fibrosis (CF).16 Finally,
1.3 equivalents of diphenylphosphoryl azide (DPPA) in the
presence of 1.1 equiv. of PPh3 and diisopropyl azodicarboxy-
the porphyrin-based tetravalent 1-DNJ inhibitor recently
described was found to selectively inhibit Golgi α-mannosidase
ManIIb (GM) over lysosomal α-mannosidase LManII (LM),
late (DIAD) at room temperature in dry THF,25 which furnished
the 6-azido intermediate 4 in 83% yield (Scheme 1). Confir-
mation of the occurred inversion of configuration at C-6 was
thus representing
chemotherapy.8
a
promising compound for cancer
given by 1D NOESY correlations between H-6 and H-1 and
between H-6 and H-3 observed for compound 4. Catalytic
hydrogenation in acidic MeOH followed by basic treatment
furnished (6S)-6-aminohyacinthacine A2 (5) in quantitative
yield (Scheme 1).
All these results illustrate the relatively newly born interest
of the scientific community in this field17 and the importance
of building new and diversified multivalent iminosugars in the
search for more selective glycosidase inhibitors and/or more
active molecular chaperones.
With the “6-epi-6,7-dideoxycasuarine like” azide 4 in hand,
we tested the CuAAC cycloaddition in the presence of alkynes
6 bearing different substituents (Table 1). The reaction was
performed in a 2 : 1 THF–H2O mixture with 0.3 equivalents of
CuSO4 and 0.6 equivalents of sodium ascorbate,26 and readily
afforded after 2–3 hours at room temperature the 1,4-disubsti-
tuted triazole adducts 7a–e in excellent yields after purification
over silica gel.27 Only in the case of glucosyl alkyne 6f, syn-
thesized in three steps from commercially available tetrabenzy-
lated glucose,28 no conversion of the starting material was
observed at room temperature, and the reaction was success-
fully performed in a MW reactor at 80 °C for 45 minutes.
Under these conditions, the triazole glucosyl adduct 7f
was obtained in 86% yield after isolation by flash column
chromatography (FCC) (Table 1).
Due to our experience in the synthesis of pyrrolizidine alka-
loids and related structural analogues,18 and based on our
recent findings that hyacinthacine and casuarine derivatives
(Fig. 1) are tight inhibitors of glycosidases of therapeutic inter-
est (e.g. human maltase glucoamylase)19 or with agrochemical
potential application (e.g., trehalases),20 we report in this work
the synthesis of the per-O-benzyl protected and unprotected
6-azido-6-epi-6,7-dideoxycasuarine key intermediates 4 and 31,
exploiting the cyclic polyhydroxylated nitrone cycloaddition
chemistry of nitrone 1.21 These compounds, prepared by
stereoselective installation of an azido moiety at C-6, allowed a
straightforward and versatile functionalization of the pyrrolizi-
dine skeleton by the CuI-catalyzed azide–alkyne cycloaddition
(CuAAC)22,23 and the preparation of the first examples of multi-
valent (3 to
9 pyrrolizidine units) 6-epi-6,7-dideoxycasu-
arine-like pyrrolizidine iminosugars. Preliminary biological
Scheme 1 Synthesis of azido intermediate 4 and of (6S)-6-amino-
hyacinthacine A2 (5).
Fig. 1 Iminosugar pyrrolizidine glycosidase inhibitors.
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Table 1 The CuAAC cycloaddition of 4 with alkynes 6
Scheme 3 Synthesis of the trivalent pyrrolizidine 10.
Entry
Alkyne
Temp. (°C)
Time
Product
Yielda (%)
Successively, the synthesis of multivalent pyrrolizidine
structures was targeted by employing different scaffolds
bearing terminal alkyne moieties. The CuAAC reaction of com-
pound 4 (3.1 equivalents) with commercially available tri-
propargyl amine (9) performed with CuSO4 (0.3 equiv.)/sodium
ascorbate (0.6 equiv.) in THF–H2O 2 : 1 in a MW reactor at
80 °C for 45 minutes31 gave a very clean crude mixture, from
which the trivalent pyrrolizidine compound 10 was isolated in
99% yield after flash column chromatography (Scheme 3).
However, catalytic hydrogenation of 10 in acidic MeOH was
troublesome, and all attempts to isolate the pure debenzylated
compound failed.32 Despite the advantage of commercial avail-
ability of this scaffold, the problems in the deprotection step
prompted us to turn our attention to other polyfunctionalized
alkynes on which our azide 4 is ligated.
1
2
3
4
5
6
6a
6b
6c
6d
6e
6f
25
25
25
25
25
80b
2 h
2 h
2 h
3 h
3 h
45 min
7a
7b
7c
7d
7e
7f
83
81
80
81
80
86
a Isolated yield after FCC. b Reaction performed in a MW reactor.
The reaction of 4 equivalents of 4 with tetravalent scaffold
11, synthesized by propargylation of pentaerythritol with pro-
pargyl bromide and NaH as previously reported,33 afforded the
expected tetravalent iminosugar derivative 12 in 91% yield
(Scheme 4). Deprotection of the perbenzylated adduct 12 was
not a trivial task, due to the high basicity and hydrophilicity of
the deprotected compound. Catalytic hydrogenation of 12 in
acidic MeOH with Pd/C gave a mixture of hydrochloride salts
that were passed onto an ion exchange resin Dowex 50WX8-
200 eluting successively with MeOH, H2O and 6% aqueous
ammonia. However, part of the compound was eluted in the
first fraction with MeOH as hydrochloride salt, and part with
ammonia in the final fraction as free amine. After treatment of
the methanolic fraction with strongly basic resin Ambersep
900 OH, pure 13 was obtained in 89% overall yield (Scheme 4).
With the aim of exploring the role of the tether in imposing
a different spatial arrangement of the multivalent iminosugar,
we synthesized a trivalent ligand with a longer alkyl chain.
Pentane-1,5-diol (14) was therefore propargylated with
propargyl bromide and NaH, affording alcohol 15 in 60% yield
(Scheme 5).34
Scheme 2 Deprotection of the adducts. Synthesis of pyrrolizidine
triazoles 8.
Catalytic hydrogenation with Pd/C in acidic MeOH afforded
pyrrolizidine derivatives 8a–d and 8f in excellent yields
(Scheme 2). Unfortunately, the acetal protecting group for the
aldehyde moiety in 7e did not tolerate these acidic conditions,
and catalytic hydrogenation afforded compounds 8e′ and 8e″
in 50% and 24% yields, respectively (Scheme 2). Compounds
8, obtained as hydrochloride salts after catalytic hydrogen-
ation, were obtained as pure free amines either by treatment
with ion exchange resin Dowex 50WX8-200 or by FCC (see the
Experimental section). Compound 8f represents an interesting
new example of an aza-C-disaccharide mimic29 in which the
iminosugar is linked to a common sugar by a non-hydrolyzable
bio-compatible triazole linkage.30
Tosylation of the primary alcohol gave tosylate 16 in 92%
yield, and final treatment with pentaerythritol (6 equivalents
of 16) and NaH gave the trivalent scaffold 17 in 71% yield
instead of the expected tetravalent compound. With this novel
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Scheme 4 Synthesis of the tetravalent pyrrolizidine 12–13.
Scheme 6 Synthesis of the trivalent pyrrolizidines 19 and 20.
Scheme 5 Synthesis of the trivalent scaffold 17.
compound in hand, the CuAAC performed as usual and
readily afforded trivalent pyrrolizidine 18 in 81% yield
(Scheme 6). After catalytic hydrogenation in acidic MeOH, the
crude mixture was directly reacted with excess of acetic
anhydride in pyridine affording peracetylated compound 19 in
77% over two steps. Final treatment with strongly basic
resin Ambersep 900 OH in MeOH overnight gave pure 20 in
quantitative yield (Scheme 6).
Propargylation of glycerol with NaH and propargyl bromide
gave 21, another good and inexpensive trivalent scaffold35 to
be used in the CuAAC reaction. The reaction of 4 (3.3 equi-
valents) with 21 performed as described above afforded the
Scheme 7 Synthesis of the trivalent pyrrolizidines 23 and 24.
trivalent pyrrolizidine 22 in 99% yield (Scheme 7). Catalytic
hydrogenation with Pd/C in acidic MeOH followed by treat-
ment with excess of acetic anhydride and pyridine at room
temperature yielded the peracetylated compound 23 in 54%
yield over two steps. Reaction of 23 with strongly basic Amber-
sep 900 OH in MeOH overnight gave compound 24 that
needed further purification by size exclusion chromatography
to yield pure 24 in 55% overall yield (Scheme 7). It should be
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Scheme 8 Synthesis of the dendrimeric nonavalent scaffold 27.
noted that peracetylated compounds 19 and 23 are also inter-
esting for biological applications, in particular for use as
pharmacological chaperones, since they could act as pro-drugs
in vivo by facilitating cellular uptake, as recently reported.15
The possibility of generating higher valent pyrrolizidine
iminosugars by employing a dendrimeric scaffold was finally
investigated. Tris[(propargyloxy)methyl]aminomethane (25),
which was prepared in a three step sequence from tris (hydroxy-
methyl)aminomethane as previously reported,36 was reacted
with trimesoyl chloride (26), in turn synthesized from trimesic
acid,37 in the presence of DIPEA in CH2Cl2 at room tempera-
ture, affording dendrimeric nonavalent scaffold 27 in 68%
yield (Scheme 8).38
Scheme 9 Synthesis of the nonavalent pyrrolizidine derivative 28.
CuAAC reaction of 27 with 9 equivalents of 4 gave nonava-
lent pyrrolizidine 28 in 91% yield (Scheme 9). However,
attempted deprotection of the benzyl groups of 28 failed.
Indeed, catalytic hydrogenation in acidic MeOH afforded a
mixture of salts that were treated with excess Ac2O and Py
affording the peracetylated derivative, which decomposed
either upon treatment with the strongly basic resin Ambersep
900 OH or with the less basic Amberlyst A21, due to the labile
amide bond.39
Scheme 10 Synthesis of the intermediate azide 31.
Catalytic hydrogenation of 29 performed as usual cleanly
gave compound 30 in 90% yield. Subsequent reaction with
NaN3 in DMF at 80 °C afforded azide 31 in 95% yield. Finally,
the reaction of dendrimeric nonavalent scaffold 27 with 9
equivalents of azide 31 yielded pure nonavalent pyrrolizidine
32 in 55% yield after purification by FCC (Scheme 11).
Preliminary biological evaluation of these new pyrrolizidine
derivatives and multivalent pyrrolizidine iminosugars was
carried out by measuring their enzyme inhibitory activity.
Compounds 5, 8a–f, 13, 20, 24, 31 and 32 as well as 6-epi-7-
deoxycasuarine were evaluated towards a panel of eleven com-
mercially available glycosidases.41 The preparation and bio-
logical evaluation as an enzyme inhibitor of 6-epi-7-
deoxycasuarine has been reported by Behr.42 Nevertheless, for
comparative purpose, we have evaluated it under the same
conditions as the other compounds.
The assessed compounds did not show inhibition towards
β-galactosidase from Escherichia coli or from Aspergillus oryzae
at 1 mM concentration and under optimal pH. Table 2 shows
the inhibition results towards α-L-fucosidase from bovine
kidney, α-galactosidase from coffee beans, α-glucosidase from
Taking into account the relatively low stability of the dendri-
meric nonavalent pyrrolizidine 28, performing the click reac-
tion as the last step was envisioned as a better strategy. To this
aim, pyrrolizidine azide with free hydroxy groups was needed
as the key precursor. This turned out to be quite troublesome,
since catalytic hydrogenation of 4 or treatment with BCl3 not
only removed the benzyl groups but also reduced the azide
moiety to the corresponding amine, while an oxidative clea-
40
vage with Na2S2O4 and NaBrO3 aimed to preserve the azido
group resulted in decomposition of the compound. Therefore,
a different strategy was considered, with introduction of the
azido moiety after deprotection of the benzyl groups via a
mesylate precursor. Alcohol 3 was then reacted with MsCl in
CH2Cl2 in the presence of NEt3 to afford mesylate 29 in 83%
yield (Scheme 10).
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interesting structure–activity relationships can be derived from
their enzyme inhibitory data. The comparison of the inhibitory
properties towards amyloglucosidases of 6-epi-7-deoxy-casuar-
ine (IC50 = 2.5 μM, Ki = 2.8 μM) and compound 5 (IC50
=
15.3 μM, Ki 18.2 = μM) indicates that the replacement of a
hydroxy with an amino group at position 6 of the pyrrolizidine
framework is detrimental, while this molecule gains moderate
inhibition towards β-N-acetylglucosaminidase (Table 2, entries
1 and 2). However, the presence of an azido group at position
6 of the bicyclic iminosugar, as in 31, improves the inhibition
towards amyloglucosidases (IC50 = 3.4 μM, Ki = 3.4 μM)] and
becomes similar to 6-epi-7-deoxycasuarine (Table 2, entries 1
and 3). When a triazole linker is attached to position 6, a
slight improvement in the inhibitory activity towards amylo-
glucosidases is detected (entries 4–10). The substitution at the
triazole linker has little effect on the inhibitory properties
against the same enzyme, given that compounds 8a–f (IC50
=
1.7–2.6 μM) present similar values and behaviour. Only
the hexyl derivative 8a shows additionally a weak-to-moderate
inhibition towards β-N-acetylglucosaminidase.
Scheme 11 Synthesis of the nonavalent pyrrolizidine 32.
Concerning the multivalent structures, the trimeric struc-
ture 24 showed similar inhibitory potency towards amylo-
glucosidase (IC50 = 1.6 μM, Ki = 2.4 μM) compared to the most
similar monovalent triazole 8e″ (Table 2, entries 9 and 13).
The trimeric tethered compound 20 presented even worse
inhibitory properties, thus indicating that the length of the
spacer has a detrimental effect on the inhibitory values; the
arms in the conjugate should move away the pyrolizidine moi-
eties from the active site. The tetrameric structure 13 pre-
sented only a three-to-fourfold increase on the inhibitory
properties towards amyloglucosidase with reference to the
monovalent triazole 8e″ (Table 2, entries 9 and 11). Thus, an
rice and yeast (Saccharomyces cerevisiae), amyloglucosidase
from Aspergillus niger, β-glucosidase from almonds, α-manno-
sidase from jack beans, β-mannosidase from snail and β-N-
acetylglucosaminidase from jack beans. It is worth mentioning
that most of the new compounds showed good, competitive
(see ESI† for Lineweaver–Burk plots) and specific inhibitory
activity towards amyloglucosidase in the low μM range. These
results are in agreement with those given by other stereo-
chemically related pyrrolizidine alkaloids. Nevertheless some
Table 2 Inhibitory activities of 6-epi-7-deoxycasuarine and compounds 5, 8(a–f), 13, 20, 24, 31 and 32 towards glycosidases. Percentage of inhi-
bition at 1 mM, IC50 (in parenthesis, μM) and Ki (bold, μM) if measured. Optimal pH, 37 °Ca,b,c
Enzymes
α-L-
β-N-
Entry Compounds
Fuc-ase α-Gal-ase α-Glc-ase Amylogluc-ase β-Glc-ase α-Man-ase β-Man-ase Acetylglucosaminidase
1
2
3
4
5
6
7
8
6-epi-7-Deoxycasuarine
5
—
—
—
—
23
—
—
—
—
20
—
—
—
21
—
—
—
—
—
—
—
—
—
—
—
29
—
49
44d
—
—
—
—
—
—
—
—
—
—
—
—
51f
98 (2.5) 2.8
98 (15.3) 18.2
98 (3.4) 3.4
98 (1.7) 2.2
98 (2.3) 3.7
99 (2.0)
98 (1.9) 4.5
97 (2.1) 1.9
96 (2.3) 2.5
99 (2.6)
—
24
39
—
—
—
—
—
—
—
—
—
—
—
—
—
39
—
—
—
—
—
—
—
—
16
26
—
—
—
20
—
—
—
—
—
—
17
—
—
—
—
—
44
17
51
19
—
—
—
—
21
—
—
—
—
31
8a
8b
8c
8d
8e′
8e″
8f
13
20
24
32
9
10
11
12
13
14
94 (1.5) 0.7
83
97 (1.6) 2.4e
97 (0.7) 0.7
a For conditions of measurements see ref. 41 and the Experimental section. b — No inhibition was detected at 1 mM concentration of the
corresponding compound. c α-L-Fucosidase from bovine kidney, α-galactosidase from coffee beans, α-glucosidase from yeast (Saccharomyces
cerevisiae), and rice, Amyloglucosidase from Aspergillus niger, β-glucosidase from almonds, α-mannosidase from jack beans, β-mannosidase from
snail and β-N-acetylglucosaminidase from jack beans. d α-Glucosidase from yeast (Saccharomyces cerevisiae). e Mixed inhibition: the Ki value
corresponding to the competitive term is given, and the non-competitive inhibition has an apparent inhibition constant Ki′ = 7.6 μM.
f α-Glucosidase from rice.
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inhibitory multivalent effect is not observed, because an 0.25 mm silica gel plates (Merck F254). Column chromato-
increase in the inhibitory potency is not over the statistical graphies were carried out on Silica Gel 60 (32–63 μm) or on silica
value when compared with the related monovalent compound. gel (230–400 mesh, Merck). Yields refer to spectroscopically
The selectivity observed in monovalent triazole 8e″ is main- and analytically pure compounds unless otherwise stated.
tained on the tetramer 13. Nonaderivative 32 displayed the 1H NMR spectra were recorded on a Varian Mercury-400 or on
same inhibitory value against amyloglucosidase than 13, a Varian INOVA 400 instrument at 25 °C. 13C NMR spectra were
although lower selectivity is observed since new weak-to-mod- recorded on a Varian Gemini-200 or on a Varian Gemini-300.
erate inhibition is found against α-galactosidase (49%) and Chemical shifts are reported relative to TMS (1H: δ = 0.00 ppm)
α-glucosidase (51%), which was not present in 13, or in any of and CDCl3 (13C: δ = 77.0 ppm). Integrals are in accordance
the monovalent compounds.
with assignments, coupling constants are given in Hz. For
detailed peak assignments 2D spectra were measured (COSY,
HSQC, NOESY, and NOE as necessary). Small scale microwave-
assisted syntheses were carried out in a CEM Discover micro-
wave apparatus for synthesis with an open reaction vessel and
an external surface sensor. IR spectra were recorded with a BX
FT-IR Perkin-Elmer System spectrophotometer. ESI-MS spectra
were recorded with a Thermo Scientific™ LCQ Fleet Ion Trap
Mass Spectrometer. Elemental analyses were performed with a
Perkin-Elmer 2400 analyzer. Optical rotation measurements
were performed on a JASCO DIP-370 polarimeter.
Conclusions
In conclusion, the preparation of two new azido intermediates
4 and 31, in turn synthesized by 1,3-dipolar cycloaddition of a
D-arabinose derived nitrone followed by N–O ring cleavage and
stereoselective installation of an azido moiety at C-6, allowed
the preparation of the first examples of multivalent pyrrolizi-
dine iminosugars with up to 9 bioactive molecules on the
same scaffold. The exploitation of the CuAAC cycloaddition on
compound 4 also enabled the preparation of a small library of
functionalized pyrrolizidines possessing the triazole ring at
C-6. The data from the biological evaluation of these com-
pounds revealed interesting results regarding inhibition
towards amyloglucosidase.
First of all, it is worth mentioning that activity towards this
enzyme is not lost despite the complexity and variety of the
new molecules tested. In the monovalent pyrrolizidines
studied, the presence of an azido group or a substituted tria-
zole linker determines the same effect of the inhibitory activity
towards amyloglucosidase than a hydroxyl group, while an
amino group is clearly detrimental. For the multivalent struc-
tures, no multivalent effect is observed towards amyloglucosi-
dase, the enzyme most influenced by this set of compounds.
The inhibitory properties of the tetramer 13 concur approxi-
mately with the statistical effect of binding four pyrrolizidine
moieties. The same value is obtained for nonavalent com-
pound 32. It is worth mentioning the role of the spacer in the
inhibitory potency: a longer linker, although detrimental in
the corresponding conjugate (20 vs. 24), provides structural
information about the binding of the pyrrolizidine moieties to
the enzyme active site. It is important to stress that, although
from the results presented here the existence of a multivalent
effect on amyloglucosidase inhibition cannot be deduced, the
structure–activity relationships obtained for the monovalent
and multivalent structures provide the basis for further studies
in the rising field of multivalency and the action of glycosi-
dases. Work is underway to evaluate the inhibition of these
compounds towards human glycosidases and their properties
as pharmacological chaperones towards lysosomal glycosidases.
Cycloaddition of nitrone 1 to N,N-dimethylformamide18d
A solution of nitrone 1 (600 mg, 1.44 mmol) and N,N-dimethyl-
formamide (223 μL, 2.16 mmol) in 4 mL of CH2Cl2 was stirred
at room temp. for 3 d, until TLC analysis (AcOEt–PE 3 : 1)
showed the disappearance of the starting material (Rf = 0.39)
and the appearance of a new product (Rf = 0.30). At completion
of the reaction, the solvent was removed under reduced
pressure and the crude mixture was purified by FCC (AcOEt–
PE 3 : 1) affording pure 2 (Rf = 0.30, 620 mg, 1.2 mmol, 83%
yield) as a white solid.
N–O ring cleavage of cycloadduct 2 and reduction of lactam to
alcohol 3 18d
To a solution of 2 (190 mg, 0.37 mmol) in 3 mL of a 9 : 1
CH3COOH–H2O mixture, Zn dust (97 mg, 1.47 mmol) was
added. The suspension was stirred at 65 °C for 3 h, then TLC
analysis (AcOEt–PE, 3 : 1) showed the disappearance of the
starting material (Rf = 0.30) and formation of a new product
(Rf = 0.44). After filtration through cotton the mixture was con-
centrated at reduced pressure and saturated aqueous solution
of NaHCO3 was added at 0 °C until basic pH was attained.
After extraction with AcOEt (3 × 25 mL), the organic layers were
dried over Na2SO4 and concentrated at reduced pressure
affording pure lactam as a white solid that was dissolved in
10 mL of dry THF and, under a nitrogen atmosphere at 0 °C,
LiAlH4 (1 m solution in THF, 1.22 mL, 1.22 mmol) was added
dropwise. The mixture was raised to room temp. and heated at
reflux temperature for 2 h, until TLC analysis (AcOEt–PE, 3 : 1)
showed the disappearance of the starting material (Rf = 0.37)
and the formation of a new product (Rf = 0.00). The reaction
was then quenched at 0 °C with a saturated aqueous solution
of Na2SO4. After extraction with AcOEt (3 × 20 mL), the organic
layers were dried on Na2SO4 and concentrated at reduced
Experimental section
Commercial reagents were used as received. All reactions were pressure affording pure 3 (161 mg, 0.35 mmol, 95% yield over
carried out under magnetic stirring and monitored by TLC on two steps) as a white solid.
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(1R,2R,3R,6S,7aR)-1,2-Bis(benzyloxy)-3-[(benzyloxy)methyl]-
6-azido-hexahydro-1H-pyrrolizidine (4)
C-2), 69.4 (d, C-3), 65.1 (d, C-7a), 62.9 (t, C-8), 60.9 (t, C-5), 49.4
(d, C-6), 37.7 (t, C-7); MS (ESI): m/z calcd for C8H16N2O3 + H+
189.12 [M + H]+; found: 189.09; elemental analysis calcd (%)
for C8H16N2O3 (188.22): C 51.05, H 8.57, N 14.88; found:
C 51.66, H 8.98, N 15.08.
To a solution of 3 (666 mg, 1.45 mmol) in dry THF (6 mL)
under a nitrogen atmosphere triphenylphosphine (422 mg,
1.60 mmol) was added. The reaction mixture was cooled to
0 °C and DIAD (315 µL, 1.60 mmol) was added dropwise,
forming a yellow precipitate. After addition of diphenylphos-
phorilazide (520 mg, 1.89 mmol), the suspension was raised
to room temp. and stirred under a nitrogen atmosphere for
2 h, until TLC analysis (CH2Cl2–MeOH 10 : 1) showed the dis-
appearance of the starting material (Rf = 0.22) and formation
of a new product (Rf = 0.95). The solvent was removed under
reduced pressure and the crude was purified by FCC
General procedure for the synthesis of 1,4-disubstituted
triazole adducts 7a–f
To a solution of 4 (1 equiv.) in a 2 : 1 THF–H2O mixture were
added CuSO4 (30 mol%), sodium ascorbate (60 mol%) and
alkyne 6a–f (1 equiv.). The reaction mixture was stirred at room
temp43 until TLC analysis (EP–AcOEt 3 : 1) showed the dis-
appearance of the starting material (Rf = 0.49) and formation
of the desired product. After filtration through Celite®, the
solvent was removed under reduced pressure and the crude
was purified by FCC to obtain the 1,4-disubstituted triazole
adducts 7a–f.
(1R,2R,3R,6S,7aR)-1,2-Bis(benzyloxy)-3-[(benzyloxy)methyl]-
6-[(4-hexyl)-1H-1,2,3-triazol-1-yl]-hexahydro-1H-pyrrolizidine
(7a). Obtained as a colorless oil in 83% yield on 0.12 mmol of
alkyne 6a after 2 h. Rf = 0.14 (PE–AcOEt 3 : 1); [α]2D6 = +8.99 (c =
1.48 in CHCl3); 1H-NMR (400 MHz, CDCl3): δ = 7.41 (s, 1H,
H-triazole), 7.36–7.25 (m, 15H, H-Ar), 5.29–5.23 (m, 1H, H-6),
4.70–4.59 (AB system, J = 11.7 Hz, 2H, H-Bn), 4.54–4.48 (AB
system, J = 11.7 Hz, 2H, H-Bn), 4.53 (s, 2H, H-Bn), 4.08 (dd, J =
5.9, 5.0 Hz, 1H, H-2), 3.90 (t, J = 5.0 Hz, 1H, H-1), 3.66 (td, J =
7.7, 5.0 Hz, 1H, H-7a), 3.60 (dd, J = 9.4, 5.3 Hz, 1H, Ha-8), 3.52
(dd, J = 9.4, 6.4 Hz, 1H, Hb-8), 3.38 (d, J = 6.0 Hz, 2H, H-5),
3.13 (q, J = 6.0 Hz, 1H, H-3), 2.68 (t, J = 7.8 Hz, 2H,
CH2CHvCH), 2.35 (ddd, J = 13.4, 7.3, 3.5 Hz, 1H, Ha-7),
2.25–2.18 (m, 1H, Hb-7), 1.65 (pquint, J = 7.6 Hz, 2H,
CH2CH2CHvCH), 1.40–1.25 (m, 6H, CH2), 0.88 (t, J = 6.9 Hz,
3H, CH3); 13C-NMR (50 MHz, CDCl3): δ = 148.2 (s, C-triazole),
138.0, 137.7, 137.4 (s, C-Ar), 128.1–127.2 (d, 15C, C-Ar), 118.9
(d, C-triazole), 88.1 (d, C-1), 85.5 (d, C-2), 73.0 (t, C-Bn), 72.1 (t,
2C, C-Bn, C-8), 71.7 (t, C-Bn), 68.8 (d, C-3), 66.5 (d, C-7a), 61.1
(d, C-6), 60.1 (t, C-5), 37.9 (t, C-7), 31.3 (t, CH2), 29.2 (t,
(PE–AcOEt 4 : 1) affording pure
4 (Rf = 0.21, 582 mg,
1.20 mmol, 83% yield) as a yellow oil. [α]2D7 = +3.96 (c = 1.06 in
CHCl3); 1H-NMR (400 MHz, CDCl3): δ = 7.30–7.18 (m, 15H,
H-Ar), 4.60–4.48 (AB system, J = 11.7 Hz, 2H, H-Bn), 4.50–4.41
(AB system, J = 11.7 Hz, 2H, H-Bn), 4.48–4.42 (AB system, J =
12.2 Hz, 2H, H-Bn), 4.10–4.06 (m, 1H, H-6), 3.96 (dd, J = 6.3,
5.4 Hz, 1H, H-2), 3.76 (t, J = 4.9 Hz, 1H, H-1), 3.59–3.54 (m, 1H,
H-7a), 3.50 (dd, J = 9.2, 5.3 Hz, 1H, Ha-8), 3.43 (dd, J = 9.2,
6.3 Hz, 1H, Hb-8), 3.07 (dd, J = 11.7, 3.4 Hz, 1H, Ha-5), 2.98
(dd, J = 11.7, 5.4 Hz, 1H, Hb-5), 2.93 (q, J = 6.3 Hz, 1H, H-3),
1.96 (ddd, J = 13.2, 6.8, 3.4 Hz, 1H, Ha-7), 1.81 (ddd, J = 13.6,
8.3, 6.3 Hz, 1H, Hb-7); 13C-NMR (50 MHz, CDCl3): δ = 138.4,
138.2, 137.9 (s, C-Ar), 128.4–127.5 (d, 15C, C-Ar), 88.3 (d, C-1),
85.9 (d, C-2), 73.3 (t, C-Bn), 72.4 (t, 2C, C-Bn, C-8), 71.9 (t,
C-Bn), 69.2 (d, C-3), 66.7 (d, C-7a), 62.1 (d, C-6), 59.9 (t, C-5),
37.0 (t, C-7); IR (CDCl3): ν = 3081, 3064, 3032, 2926, 2864,
2102, 1496, 1453, 1365, 1264, 1099 cm−1; MS (ESI): m/z calcd
for C29H32N4O3 + H+ 485.25 [M + H]+; found: 485.33; elemental
analysis calcd (%) for C29H32N4O3 (484.59): C 71.88, H 6.66,
N 11.56; found: C 71.45, H 6.54, N 11.68.
(1R,2R,3R,6S,7aR)-6-Amino-3-hydroxymethylhexahydro-1H-
pyrrolizidine-1,2-diol (5)
To a solution of 4 (112 mg, 0.23 mmol) in 10 mL of methanol CH2CH2CHvCH), 28.7 (t, CH2), 25.5 (t, CH2CHvCH), 22.3 (t,
56 mg of 10% Pd/C and two drops of 37% HCl were added CH2), 13.8 (q, CH3); IR (CDCl3): ν = 3088, 3065, 3031, 2928,
under a nitrogen atmosphere, and then the mixture was stirred 2859, 1708, 1454, 1361, 1223, 1110, 1094 cm−1; MS (ESI): m/z
under a hydrogen atmosphere at room temp. for 1 day. TLC calcd for C37H46N4O3 + H+ 595.36 [M + H+]; found: 595.43;
analysis (PE–AcOEt 3 : 1) showed the disappearance of the elemental analysis calcd (%) for C37H46N4O3 (594.79): C 74.72,
starting material (Rf = 0.35) and formation of a new product H 7.80, N 9.42; found: C 74.40, H 8.03, N 8.95.
(Rf = 0.00). The mixture was filtered through Celite® and the
(1R,2R,3R,6S,7aR)-1,2-Bis(benzyloxy)-3-[(benzyloxy)methyl]-
solvent was removed under reduced pressure affording a crude 6-[4-(2-hydroxyethyl)-1H-1,2,3-triazol-1-yl]-hexahydro-1H-
yellow oil (117 mg). Free amine was obtained by passing the pyrrolizidine (7b). Obtained as a colorless oil in 81% yield on
hydrochloride through a Dowex 50WX8 ion-exchange resin. 0.09 mmol of alkyne 6b after 2 h. Rf = 0.36 (CH2Cl2–MeOH
Elution with 6% NH4OH afforded the free base 5 (43 mg, 12 : 1); [α]2D5 = +14.0 (c = 0.92 in CHCl3); 1H-NMR (400 MHz,
0.23 mmol, 100% yield over two steps) as a waxy brown solid. CDCl3): δ = 7.53 (s, 1H, H-triazole), 7.35–7.26 (m, 15H, H-Ar),
1
[α]2D3 = +16.7 (c = 1.01 in MeOH); H-NMR (400 MHz, D2O): δ = 5.30–5.23 (m, 1H, H-6), 4.71–4.56 (AB system, J = 11.7 Hz, 2H,
3.66–3.59 (m, 3H, H-1, H-2, Ha-8), 3.49 (dd, J = 11.7, 6.3 Hz, H-Bn), 4.54–4.47 (AB system, J = 11.7 Hz, 2H, H-Bn), 4.52 (s,
1H, Hb-8), 3.46–3.40 (m, 1H, H-6), 3.15 (td, J = 7.8, 3.9 Hz, 1H, 2H, H-Bn), 4.08 (dd, J = 5.9, 5.0 Hz, 1H, H-2), 3.96–3.92 (m, 2H,
H-7a), 2.85 (dd, J = 11.7, 6.3 Hz, 1H, Ha-5), 2.59 (ddd, J = 9.8, CH2OH), 3.90 (t, J = 5.0 Hz, 1H, H-1), 3.65 (td, J = 7.9, 5.3 Hz,
6.3, 3.9 Hz, 1H, H-3), 2.47 (dd, J = 11.7, 7.8 Hz, 1H, Hb-5), 1.96 1H, H-7a), 3.59 (dd, J = 9.4, 5.3 Hz, 1H, Ha-8), 3.51 (dd, J = 9.4,
(ddd, J = 12.7, 6.4, 3.9 Hz, 1H, Ha-7), 1.62 (dt, J = 12.7, 8.3 Hz, 6.4 Hz, 1H, Hb-8), 3.40–3.38 (m, 2H, H-5), 3.14 (q, J = 6.2 Hz,
1H, Hb-7); 13C-NMR (50 MHz, D2O): δ = 80.3 (d, C-1) 76.6 (d, 1H, H-3), 2.92 (t, J = 5.9 Hz, 2H, CH2CH2OH), 2.68 (bs, 1H,
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OH), 2.36 (ddd, J = 13.4, 7.3, 3.5 Hz, 1H, Ha-7), 2.22 (ddd, J = 1326, 1262, 1240, 1208, 1100 cm−1; MS (ESI): m/z calcd for
13.5, 7.9, 6.8 Hz, 1H, Hb-7); 13C-NMR (50 MHz, CDCl3): δ = C33H36N4O5 + H+ 569.27 [M + H+]; found: 569.38; elemental
145.6 (s, C-triazole), 138.3, 138.0, 137.7 (s, C-Ar), 128.5–127.6 analysis calcd (%) for C33H36N4O5 (568.66): C 69.70, H 6.38, N
(d, 15C, C-Ar), 120.3 (d, C-triazole), 88.4 (d, C-1), 85.9 (d, C-2), 9.85; found: C 69.21, H 5.98, N 10.17.
73.3 (t, C-Bn), 72.4 (t, C-Bn), 72.3 (t, C-Bn), 72.0 (t, C-8), 69.1
(1R,2R,3R,6S,7aR)-1,2-Bis(benzyloxy)-3-[(benzyloxy)methyl]-
(d, C-3), 66.8 (d, C-7a), 61.6 (d, C-6), 61.5 (t, CH2OH), 60.4 6-[(4-diethoxymethyl)-1H-1,2,3-triazol-1-yl]-hexahydro-1H-
(t, C-5), 38.1 (t, CH2CH2OH), 28.7 (t, C-7); IR (CDCl3): ν = 3622, pyrrolizidine (7e). Obtained as a colorless oil in 80% yield on
3469, 3088, 3066, 3032, 2865, 2247, 1496, 1453, 1365, 1112, 0.11 mmol of alkyne 6e after 3 h. Rf = 0.28 (PE–AcOEt 1 : 1);
1
1062 cm−1; MS (ESI): m/z calcd for C33H38N4O4 + H+ 555.29 [α]D23 = +9.2 (c = 0.9 in CHCl3); H-NMR (400 MHz, CDCl3): δ =
[M + H+]; found: 555.40; elemental analysis calcd (%) for 7.65 (s, 1H, H-triazole), 7.28–7.18 (m, 15H, H-Ar), 5.63 (s, 1H,
C33H38N4O4 (554.68): C 71.46, H 6.91, N 10.10; found: C 71.06, CH(OCH2CH3)2), 5.22–5.17 (m, 1H, H-6), 4.54–4.51 (AB system,
H 6.98, N 10.19.
J = 11.7 Hz, 2H, H-Bn), 4.47–4.41 (m, 4H, H-Bn), 4.00 (dd, J =
(1R,2R,3R,6S,7aR)-1,2-Bis(benzyloxy)-3-[(benzyloxy)methyl]- 5.8, 4.9 Hz, 1H, H-2), 3.83 (t, J = 4.9 Hz, 1H, H-1), 3.67–3.50 (m,
6-[(4-phenyl)-1H-1,2,3-triazol-1-yl]-hexahydro-1H-pyrrolizidine 6H, Ha-8, H-7a, CH(OCH2CH3)2), 3.44 (dd, J = 9.3, 6.3 Hz, 1H,
(7c). Obtained as colorless oil in 80% yield on 0.11 mmol of Hb-8), 3.33 (d, J = 5.4 Hz, 2H, H-5), 3.06 (q, J = 5.8 Hz, 1H,
alkyne 6c after 2 h. Rf = 0.28 (PE–AcOEt 2 : 1); [α]2D5 = +5.33 (c = H-3), 2.29 (ddd, J = 13.6, 7.3, 3.9 Hz, 1H, Ha-7), 2.20–2.13 (m,
1
1.05 in CHCl3); H-NMR (400 MHz, CDCl3): δ = 7.92 (s, 1H, H- 1H, Hb-7), 1.17 (t, J = 7.1 Hz, 3H, CH3), 1.16 (t, J = 7.1 Hz, 3H,
triazole), 7.83–7.80 (m, 2H, H-Ar), 7.43–7.39 (m, 2H, H-Ar), CH3); 13C-NMR (50 MHz, CDCl3): δ = 147.2 (s, C-triazole),
7.36–7.26 (m, 15H, H-Ar), 7.25 (s, 1H, H-Ar), 5.35 (dq, J = 6.4, 138.3, 138.1, 137.7 (s, C-Ar), 128.5–127.6 (d, 15C, C-Ar), 120.6
3.5 Hz, 1H, H-6), 4.71–4.60 (AB system, J = 11.7 Hz, 2H, H-Bn), (d, C-triazole), 97.0 (d, CH(OCH2CH3)2), 88.4 (d, C-1), 85.9 (d,
4.58–4.52 (AB system, J = 11.7 Hz, 2H, H-Bn), 4.54–4.48 (AB C-2), 73.3 (t, C-Bn), 72.4 (t, 2C, C-Bn, C-8), 72.0 (t, C-Bn), 69.1
system, J = 11.7 Hz, 2H, H-Bn), 4.11 (dd, J = 5.9, 4.9 Hz, 1H, (d, C-3), 66.8 (d, C-7a), 61.8 (t, 2C, CH(OCH2CH3)2), 61.5 (d,
H-2), 3.93 (t, J = 4.9 Hz, 1H, H-1), 3.70 (td, J = 7.8, 4.9 Hz, 1H, C-6), 60.4 (t, C-5), 38.1 (t, C-7), 15.2 (q, 2C, CH3); IR (CDCl3):
H-7a), 3.62 (dd, J = 9.3, 5.4 Hz, 1H, Ha-8), 3.54 (dd, J = 9.3, 6.4 ν = 3031, 2978, 1496, 1454, 1364, 1230, 1206, 1027, 1001 cm−1
;
Hz, 1H, Hb-8), 3.48 (dd, J = 11.7, 3.4 Hz, 1H, Ha-5), 3.42 (dd, MS (ESI): m/z calcd for C36H44N4O5 + H+ 613.33 [M + H+];
J = 11.7, 5.4 Hz, 1H, Hb-5), 3.17 (q, J = 5.9 Hz, 1H, H-3), 2.40 found: 613.45; elemental analysis calcd (%) for C36H44N4O5
(ddd, J = 13.6, 7.3, 3.4 Hz, 1H, Ha-7), 2.26 (ddd, J = 13.7, 8.3, (612.76): C 70.56, H 7.24, N 9.14; found: C 70.37, H 7.18, N
6.8 Hz, 1H, Hb-7); 13C-NMR (50 MHz, CDCl3): δ = 147.5 (s, 9.05.
C-triazole), 138.0, 137.7, 137.4 (s, C-Ar), 130.4 (s, C-Ar),
(1R,2R,3R,6S,7aR)-1,2-Bis(benzyloxy)-3-[(benzyloxy)methyl]-
128.4–127.2 (d, 19C, C-Ar), 125.4 (d, C-Ar), 118.1 (d, C-triazole), 6-[(4-tetrabenzylglucosyl)-1H-1,2,3-triazol-1-yl]-hexahydro-1H-
88.1 (d, C-1), 85.7 (d, C-2), 73.0 (t, C-Bn), 72.2 (t, C-Bn), 72.0 (t, pyrrolizidine (7f). Obtained as a colorless oil in 86% yield on
C-8), 71.8 (t, C-Bn), 68.9 (d, C-3), 66.5 (d, C-7a), 61.6 (d, C-6), 0.06 mol of alkyne 6f after heating to 80 °C in a MW reactor
60.1 (t, C-5), 38.0 (t, C-7); IR (CDCl3): ν = 3087, 3066, 3031, for 45 minutes. Rf = 0.21 (PE–AcOEt 3 : 2); [α]2D2 = +26.3 (c = 1.28
2928, 2865, 1496, 1454, 1264, 1206, 1097, 1075, 1027 cm−1. MS in CHCl3); 1H-NMR (400 MHz, CDCl3): δ = 7.73 (s, 1H, H-tri-
(ESI): m/z calcd for C37H38N4O3 + H+ 587.29 [M + H+]; found: azole), 7.34–7.18 (m, 33H, H-Ar), 7.14–7.12 (m, 2H, H-Ar), 5.26
587.42; elemental analysis calcd (%) for C37H38N4O3 (586.72): (d, J = 5.9 Hz. 1H, H-1′), 5.24–5.19 (m, 1H, H-6), 4.92 (d, J =
C 75.74, H 6.53, N 9.55; found: C 75.33, H 6.42, N 9.10.
10.7 Hz. 1H, H-Bn), 4.83–4.78 (AB system, J = 10.7 Hz, 2H,
(1R,2R,3R,6S,7aR)-1,2-Bis(benzyloxy)-3-[(benzyloxy)methyl]- H-Bn), 4.70–4.44 (m, 11H, H-Bn), 4.20–4.15 (m, 1H, H-3′), 4.04
6-[(4-methylcarboxylate)-1H-1,2,3-triazol-1-yl]-hexahydro-1H- (dd, J = 5.9, 4.9 Hz, 1H, H-2), 3.98 (dd, J = 9.2, 5.8 Hz, 1H,
pyrrolizidine (7d). Obtained as a colorless oil in 81% yield on H-2′), 3.89 (t, J = 4.9 Hz, 1H, H-1), 3.77 (d, J = 5.3 Hz, 2H, H-4′,
0.11 mmol of alkyne 6d after 3 h. Rf = 0.17 (PE–AcOEt 2 : 1); H-5′), 3.72–3.63 (m, 3H, H-7a, H-6′), 3.56 (dd, J = 9.3, 5.4 Hz,
1
[α]2D4 = +17.1 (c = 1.18 in CHCl3); H-NMR (400 MHz, CDCl3): δ 1H, Ha-8), 3.45 (dd, J = 9.3, 6.8 Hz, 1H, Hb-8), 3.43 (dd, J =
= 8.27 (s, 1H, H-triazole), 7.34–7.24 (m, 15H, H-Ar), 5.35–5.31 11.7, 5.9 Hz, 1H, Ha-5), 3.37 (dd, J = 11.7, 4.4 Hz, 1H, Hb-5),
(m, 1H, H-6), 4.67–4.57 (AB system, J = 11.7 Hz, 2H, H-Bn), 3.12 (q, J = 5.8 Hz, 1H, H-3), 2.39 (ddd, J = 13.2, 7.3, 3.9 Hz,
4.51 (s, 2H, H-Bn), 4.48 (s, 2H, H-Bn), 4.08 (dd, J = 5.5, 5.0 Hz, 1H, Ha-7), 2.25 (dt, J = 13.7, 7.3 Hz, 1H, Hb-7); 13C-NMR
1H, H-2), 3.94 (s, 3H, OMe), 3.89 (t, J = 5.0 Hz, 1H, H-1), 3.59 (50 MHz, CDCl3): δ = 143.8 (s, C-triazole), 138.8–137.8 (s, 7C,
(td, J = 8.2, 5.1 Hz, 1H, H-7a), 3.58 (dd, J = 9.4, 5.5 Hz, 1H, Ha- C-Ar), 128.5–127.6 (d, 35C, C-Ar), 122.9 (d, C-triazole), 88.5 (d,
8), 3.50 (dd, J = 9.3, 6.6 Hz, 1H, Hb-8), 3.40–3.39 (m, 2H, H-5), C-1), 85.9 (d, C-2), 82.4 (d, C-3′), 79.8 (d, C-2′), 78.0 (d, C-5′),
3.16 (q, J = 6.1 Hz, 1H, H-3), 2.30 (ddd, J = 13.7, 7.2, 2.9 Hz, 75.4–72.0 (d or t, 9C, 7 C-Bn, C-4′, C-8), 69.2 (d, C-1′), 69.1 (d,
1H, Ha-7), 2.22 (ddd, J = 13.7, 8.4, 6.6 Hz, 1H, Hb-7); 13C-NMR C-3), 68.7 (t, C-6′), 66.9 (d, C-7a), 61.1 (d, C-6), 60.4 (t, C-5),
(50 MHz, CDCl3): δ = 161.2 (s, CvO), 140.0 (s, C-triazole), 38.0 (t, C-7); IR (CDCl3):
ν = 3088, 3065, 3031, 2920,
138.3, 138.0, 137.7 (s, C-Ar), 128.5–127.6 (d, 15C, C-Ar), 126.5 2867, 1495, 1454, 1361, 1207, 1087, 1074, 1027 cm−1; MS (ESI):
(d, C-triazole), 88.4 (d, C-1), 86.1 (d, C-2), 73.3 (t, C-Bn), 72.4 (t, m/z calcd for C65H68N4O8 + H+ 1033.50 [M + H+]; found:
C-Bn), 72.3 (t, C-8), 72.1 (t, C-Bn), 69.1 (d, C-3), 66.6 (d, C-7a), 1033.57; elemental analysis calcd (%) for C65H68N4O8
62.4 (d, C-6), 60.3 (t, C-5), 52.1 (q, OMe), 38.1 (t, C-7); IR (1033.26): C 75.56, H 6.63, N 5.42; found: C 75.71, H 6.99,
(CDCl3): ν = 3088, 3066, 3031, 2951, 2864, 1730, 1496, 1454, N 5.26.
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General procedure for the synthesis of pyrrolizidine derivatives a white solid in 100% yield on 0.15 mmol of adduct 7c
8a–d and 8f
after 6 h. Rf = 0.29 (CH2Cl2–MeOH 4 : 1 + 1% v/v of
6% NH4OH); M.p. 136–137 °C; [α]2D2 = +14.8 (c = 0.27 in
To a solution of compounds 7a–d and 7f (1 equiv.) in metha- MeOH); 1H-NMR (400 MHz, CD3OD): δ = 8.52–8.50 (m, 1H,
nol, 10% Pd/C (50% weight) and two drops of 37% HCl were H-triazole), 7.83 (d, J = 7.8 Hz, 2H, H-Ar), 7.43 (t, J = 7.3 Hz,
added under a nitrogen atmosphere, then the mixture was 2H, H-Ar), 7.34 (t, J = 7.3 Hz, 1H, H-Ar), 5.57–5.56 (m, 1H, H-6),
stirred under a hydrogen atmosphere at room temp. At com- 4.07–3.98 (m, 3H, H-1, H-2, H-7a), 3.94 (dd, J = 12.7, 3.9 Hz,
pletion of the reaction, the mixture was filtered through 1H, Ha-5), 3.90–3.76 (m, 3H, Hb-5, H-8), 3.34–3.30 (m, 1H,
Celite® and the solvent was removed under reduced pressure H-3), 2.82–2.76 (m, 1H, Ha-7), 2.74–2.67 (m, 1H, Hb-7);
affording a crude oil. Free amines were obtained by purifi- 13C-NMR (50 MHz, CD3OD): δ = 147.8 (s, C-triazole), 130.1 (s,
cation either with the ion exchange resin Dowex 50WX8-200 C-Ar), 128.6 (d, 2C, C-Ar), 128.1 (d, C-Ar), 125.4 (d, 2C, C-Ar),
eluting successively with MeOH, H2O and 6% NH4OH 120.8 (d, C-triazole), 79.3, 77.2 (d, C-1, C-2), 74.2 (d, C-3), 70.5
(8a,8b,8f) or by FCC using mixtures of CH2Cl2–MeOH as (d, C-7a), 60.3 (d, C-6), 59.8, 59.4 (t, C-5, C-8), 35.3 (t, C-7); MS
eluents with addition (1% v/v) of 6% NH4OH (8c,8d,8e′,8e″).
(ESI): m/z calcd for C16H20N4O3 + Na+ 339.14 [M + Na]+; found:
(1R,2R,3R,6S,7aR)-6-[(4-Hexyl)-1H-1,2,3-triazol-1-yl]-3-(hydroxy- 339.30; elemental analysis calcd (%) for C16H20N4O3 (316.35):
methyl)-hexahydro-1H-pyrrolizidine-1,2-diol (8a). Obtained as C 60.75, H 6.37, N 17.71; found: C 60.37, H 6.21, N 17.58.
a pale yellow solid in 87% yield on 0.05 mmol of adduct 7a
(1R,2R,3R,6S,7aR)-6-[4-(Methylcarboxylate)-1H-1,2,3-triazol-
after 3 days. M.p. 113–115 °C; [α]2D4 = +15.8 (c = 0.55 in MeOH); 1-yl]-3-(hydroxymethyl)-hexahydro-1H-pyrrolizidine-1,2-diol
1H-NMR (400 MHz, CD3OD): δ = 7.85 (s, 1H, H-triazole), 5.30 (8d). Obtained as a waxy yellow solid in 92% yield on
(quint, J = 5.6 Hz, 1H, H-6), 3.88 (t, J = 7.3 Hz, 1H, H-2), 3.82 (t, 0.09 mmol of adduct 7d after 16 h. Rf = 0.36 (CH2Cl2–MeOH
J = 6.8 Hz, 1H, H-1), 3.75 (dd, J = 11.2, 3.4 Hz, 1H, Ha-8), 3.61 4 : 1 + 1% v/v of 6% NH4OH); [α]2D4 = +15.4 (c = 0.59 in MeOH);
(dd, J = 11.2, 5.8 Hz, 1H, Hb-8), 3.56 (q, J = 7.1 Hz, 1H, H-7a), 1H-NMR (400 MHz, CD3OD): δ = 8.65 (s, 1H, H-triazole),
3.47 (dd, J = 11.7, 4.4 Hz, 1H, Ha-5), 3.41 (dd, J = 11.7, 5.9 Hz, 5.47–5.42 (pquint., J = 4.9 Hz, 1H, H-6), 3.92–3.85 (m, 2H, H-1,
1H, Hb-5), 2.86 (ddd, J = 9.3, 6.4, 3.5 Hz, 1H, H-3), 2.67 (t, J = H-2), 3.91 (s, 3H, OMe), 3.77 (dd, J = 11.2, 3.4 Hz, 1H, Ha-8),
7.8 Hz, 2H, CH2CHvCH), 2.51 (ddd, J = 13.2, 7.8, 5.1 Hz, 1H, 3.68–3.57 (m, 3H, Hb-8, Ha-5, H-7a), 3.52–3.48 (m, 1H, Hb-5),
Ha-7), 2.41 (dt, J = 13.2, 6.8 Hz, 1H, Hb-7), 1.65 (pquint, J = 7.4 2.98–2.90 (m, 1H, H-3), 2.62–2.56 (m, 1H, Ha-7), 2.49 (dt, J =
Hz, 2H, CH2CH2CHvCH), 1.40–1.28 (m, 6H, CH2), 0.89 (t, J = 13.6, 6.8 Hz, 1H, Hb-7); 13C-NMR (50 MHz, CD3OD): δ = 160.0
6.8 Hz, 3H, CH3); 13C-NMR (50 MHz, CD3OD): δ = 148.0 (s, (s, CvO), 138.2, (s, C-triazole), 126.6 (d, C-triazole), 79.9 (d,
C-triazole), 120.8 (d, C-triazole), 81.2 (d, C-1), 77.7 (d, C-2), 71.8 C-1), 76.8 (d, C-2), 71.4 (d, C-3), 66.6 (d, C-7a), 60.9 (t, C-8),
(d, C-3), 67.0 (d, C-7a), 62.2 (t, C-8), 60.7 (d, C-6), 59.6 (t, C-5), 60.6 (d, C-6), 58.8 (t, C-5), 50.2 (q, OMe), 35.3 (t, C-7); MS (ESI):
36.5 (t, C-7), 31.3 (t, CH2), 29.1 (t, CH2CH2CHvCH), 28.5 (t, m/z calcd for C12H18N4O5 + Na+ 321.12 [M + Na]+; found:
CH2), 24.9 (t, CH2CHvCH), 22.2 (t, CH2), 12.9 (q, CH3); MS 321.16; elemental analysis calcd (%) for C12H18N4O5 (298.30):
(ESI): m/z calcd for C16H28N4O3 + Na+ 347.21 [M + Na]+; found: C 48.32, H 6.08, N 18.78; found: C 48.46, H 6.32, N 18.92.
347.28; elemental analysis calcd (%) for C16H28N4O3 (324.42):
C 59.24, H 8.70, N 17.27; found: C 59.18, H 8.48, N 17.09.
(1R,2R,3R,6S,7aR)-6-[4-(Glucosyl)-1H-1,2,3-triazol-1-yl]-3-
(hydroxymethyl) hexahydro-1H-pyrrolizidine-1,2-diol (8f).
(1R,2R,3R,6S,7aR)-6-[4-(2-Hydroxyethyl)-1H-1,2,3-triazol-1- Obtained as a waxy white solid in 91% yield on 0.04 mmol of
yl]-3-(hydroxymethyl)-hexahydro-1H-pyrrolizidine-1,2-diol (8b). adduct 7f after 1 day. [α]2D2 = +62.3 (c = 0.44 in MeOH); 1H-NMR
Obtained as a colorless oil in 90% yield on 0.05 mmol of (400 MHz, CD3OD): δ = 8.12 (s, 1H, H-triazole), 5.32 (quint, J =
adduct 7b after 3 days. [α]2D3 = +11.9 (c = 0.74 in MeOH); 5.6 Hz, 1H, H-6), 5.18 (d, J = 5.4 Hz. 1H, H-1′) 3.87–3.83 (m,
1H-NMR (400 MHz, CD3OD): δ = 7.90 (s, 1H, H-triazole), 5.29 3H, H-2, H-2′, H-3′), 3.80 (t, J = 7.1 Hz, 1H, H-1), 3.76–3.72 (m,
(quint, J = 5.4 Hz, 1H, H-6), 3.86 (t, J = 7.3 Hz, 1H, H-2), 3.79 (t, 2H, Ha-8, Ha-6′), 3.66 (dd, J = 12.2, 4.9 Hz, 1H, Hb-6′), 3.58
J = 7.3 Hz, 1H, H-1), 3.78 (t, J = 6.6 Hz, 2H, CH2OH), 3.73 (dd, (dd, J = 11.7, 6.3 Hz, 1H, Hb-8), 3.52 (q, J = 6.8 Hz, 1H, H-7a),
J = 11.2, 3.2 Hz, 1H, Ha-8), 3.58 (dd, J = 11.2, 5.9 Hz, 1H, Hb-8), 3.45–3.36 (m, 4H, H-5, H-4′ and H-5′), 2.80 (ddd, J = 8.8, 6.4,
3.50 (q, J = 7.3 Hz, 1H, H-7a), 3.41 (dd, J = 11.7, 4.8 Hz, 1H, Ha- 3.4 Hz, 1H, H-3), 2.52 (ddd, J = 13.2, 7.8, 5.4 Hz, 1H, Ha-7),
5), 3.36 (dd, J = 11.7, 5.9 Hz, 1H, Hb-5), 2.89 (t, J = 6.6 Hz, 2H, 2.40 (dt, J = 13.2, 6.6 Hz, 1H, Hb-7); 13C-NMR (50 MHz,
CH2CH2OH), 2.79 (ddd, J = 8.8, 5.8, 3.4 Hz, 1H, H-3), 2.49 CD3OD): δ = 142.8 (s, C-triazole), 122.9 (d, C-triazole), 80.4 (d,
(ddd, J = 13.2, 7.8, 5.4 Hz, 1H, Ha-7), 2.38 (dt, J = 13.2, 6.8 Hz, C-1), 76.8 (d, C-2), 74.1 (d, C-5′), 73.2 (d, C-2′), 70.6 (d, C-3),
1H, Hb-7); 13C-NMR (50 MHz, CD3OD): δ = 143.9 (s, C-triazole), 70.5 (d, C-3′), 69.9 (d, C-1′), 69.7 (d, C-4′), 65.7 (d, C-7a), 61.7 (t,
120.7 (d, C-triazole), 80.3 (d, C-1), 76.8 (d, C-2), 70.8 (d, C-3), C-8), 60.6 (d. C-6), 59.8 (t, C-6′), 58.7 (t, C-5), 35.7 (t, C-7); MS
66.0 (d, C-7a), 61.3 (t, C-8), 59.9 (d, C-6), 59.8 (t, CH2OH), 58.7 (ESI): m/z calcd for C16H26N4O8 + Na+ 425.16 [M + Na]+; found:
(t, C-5), 35.6 (t, C-7), 27.7 (t, CH2CH2OH); MS (ESI): m/z calcd 425.25; elemental analysis calcd (%) for C16H26N4O8 (402.40):
for C12H20N4O4 + Na+ 307.14 [M + Na]+; found: 307.25; elemen- C 47.76, H 6.51, N 13.92; found: C 47.89, H 6.60, N 13.48.
tal analysis calcd (%) for C12H20N4O4 (284.31): C 50.69, H 7.09,
N 19.71; found: C 50.23, H 6.97, N 19.27.
Catalytic hydrogenation of 1,4-disubstituted triazole adduct
7e. To a solution of 7f (59 mg, 0.096 mmol) in 10 mL of
(1R,2R,3R,6S,7aR)-6-[4-(Phenyl)-1H-1,2,3-triazol-1-yl]-3-(hydroxy- ethanol, 10% Pd/C (50% weight) and two drops of 37% HCl
methyl)-hexahydro-1H-pyrrolizidine-1,2-diol (8c). Obtained as were added under a nitrogen atmosphere, and then the
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mixture was stirred under a hydrogen atmosphere at room 6% NH4OH) affording pure 10 (Rf = 0.47, 157 mg, 0.1 mmol,
temp for 5 days. TLC analysis (EP–AcOEt 1 : 1) showed the dis- 99%) as a yellow oil. [α]2D2 = +6.15 (c = 0.26, CHCl3); 1H-NMR
appearance of the starting material (Rf = 0.36) and formation (400 MHz, CDCl3): δ = 7.83 (s, H-triazole), 7.70 (s, H-triazole),
of a new product (Rf = 0.00). The mixture was filtered through 7.67 (s, H-triazole), 7.32–7.24 (m, 45H, H-Ar), 5.28–5.17 (m,
Celite® and the solvent was removed under reduced pressure. 3H, H-6), 4.69–4.47 (m, 18H, H-Bn), 4.09–4.04 (m, 3H, H-2),
Then, the crude was purified by FCC (CH2Cl2–MeOH 4 : 1 + 1% 3.90–3.88 (m, 3H, H-1), 3.86, 3.84, 3.76 (s, 6H, NCH2),
v/v of 6% NH4OH) affording pure 8e′ (Rf = 0.17, 13 mg, 3.73–3.63 (m, 3H, H-7a), 3.60–3.51 (m, 6H, H-8), 3.45–3.37 (m,
0.048 mmol, 50% yield) and 8e″ (Rf = 0.23, 7 mg, 0.024 mmol, 6H, H-5), 3.16–3.09 (m, 3H, H-3), 2.45–3.32 (m, 3H, Ha-7),
24% yield) as a colorless oils.
2.30–2.19 (m, 3H, Hb-7); 13C-NMR (50 MHz, CDCl3): δ = 144.2,
(1R,2R,3R,6S,7aR)-6-[4-(Hydroxymethyl)-1H-1,2,3-triazol- 144.0, 143.5 (s, C-triazole), 138.0–137.4 (s, 9C, C-Ar),
1-yl]-3-(hydroxymethyl)-hexahydro-1H-pyrrolizidine-1,2-diol 128.1–127.2 (d, 45C, C-Ar), 122.3, 121.6, 121.3 (d, C-triazole),
(8e′). [α]2D3 = +61.2 (c = 0.26 in MeOH); 1H-NMR (400 MHz, 88.0 (d, 3C, C-1), 85.4 (d, 3C, C-2), 73.5–71.7 (t, 9C, C-Bn), 71.6
CD3OD): δ = 8.03 (s, 1H, H-triazole), 5.33 (quint, J = 5.4 Hz, 1H, (t, 3C, C-8), 68.8 (d, 3C, C-3), 66.5 (d, 3C, C-7a), 61.2, 61.0, 60.6
H-6), 4.67 (s, 2H, OCH2triazole), 3.87 (t, J = 7.8 Hz, 1H, H-2), (d, C-6), 47.9, 47.7, 47.0 (t, NCH2), 41.7 (t, 3C, C-5), 37.9, 37.7,
3.80 (t, J = 7.1 Hz, 1H, H-1), 3.74 (dd, J = 11.2, 3.4 Hz, 1H, Ha- 37.6 (t, C-7); MS (ESI): m/z calcd for C96H105N13O9 + Na+
8), 3.59 (dd, J = 11.7, 5.9 Hz, 1H, Hb-8), 3.50 (q, J = 6.8 Hz, 1H, 1606.81 [M + Na]+; found: 1606.83; elemental analysis calcd
H-7a), 3.41 (dd, J = 11.7, 4.9 Hz, 1H, Ha-5), 3.41–3.36 (m, 1H, (%) for C96H105N13O9 (1584.94): C 72.75, H 6.68, N 11.49;
Hb-5), 2.79 (ddd, J = 8.8, 5.9, 3.5 Hz, 1H, H-3), 2.49 (ddd, J = found: C 72.81, H 6.18, N 11.24.
12.9, 7.3, 4.9 Hz, 1H, Ha-7), 2.39 (dt, J = 13.4, 6.7 Hz, 1H, Hb-
Synthesis of tetravalent scaffold 11
7); 13C-NMR (50 MHz, CD3OD): δ = 147.6 (s, C-triazole), 121.8
(d, C-triazole), 81.3 (d, C-1), 77.8 (d, C-2), 71.4 (d, C-3), 66.5 (d, A solution of pentaerythritol (200 mg, 1.47 mmol) in 15 mL of
C-7a), 62.5 (t, C-8), 60.9 (d, C-6), 59.6 (t, C-5), 55.1 (t, OCH2tri- dry DMF was cooled to 0 °C and NaH (60% w/w in mineral oil,
azole), 36.6 (t, C-7); MS (ESI): m/z calcd for C11H18N4O4 + Na+ 1.53 g, 38.2 mmol) was added. After stirring for 30 min. at
293.12 [M + Na]+; found: 293.18; elemental analysis calcd (%) 0 °C, propargyl bromide (80% w/w in toluene, 1.31 g,
for C11H18N4O4 (270.29): C 48.88, H 6.71, N 20.73; found: C 8.82 mmol) was slowly added dropwise over 20 minutes. The
48.65, H 6.24, N 20.34.
(1R,2R,3R,6S,7aR)-6-[4-(Ethoxymethyl)-1H-1,2,3-triazol-1-yl]- precipitate was observed. Then, the reaction mixture was
3-(hydroxymethyl)-hexahydro-1H-pyrrolizidine-1,2-diol (8e″). stirred under a nitrogen atmosphere at room temp. for 16 h,
colour of the solution changed to brown and formation of a
[α]2D3 = +27.5 (c = 0.12 in MeOH); 1H-NMR (400 MHz, until TLC analysis (PE–AcOEt 3 : 1) showed the disappearance
CD3OD): δ = 8.08 (s, 1H, H-triazole), 5.39–5.34 (m, 1H, H-6), of the starting material (Rf = 0.00) and formation of a new
4.57 (s, 2H, OCH2triazole), 3.89 (t, J = 7.3 Hz, 1H, H-2), 3.84 (t, product (Rf = 0.59). The reaction was quenched with methanol
J = 6.9 Hz, 1H, H-1), 3.76 (dd, J = 11.2, 3.4 Hz, 1H, Ha-8), and the solvent was removed under reduced pressure. After
3.64–3.54 (m, 4H, Hb-8, H-7a, CH3CH2O), 3.53–3.42 (m, 2H, extraction with AcOEt (3 × 20 mL), the combined organic
H-5), 2.89–2.86 (m, 1H, H-3), 2.53 (ddd, J = 12.9, 7.8, 5.1 Hz, layers were dried on Na2SO4, concentrated at reduced pressure
1H, Ha-7), 2.47–2.41 (m, 1H, Hb-7), 1.19 (t, J = 7.1 Hz, 3H, and the crude mixture was purified by FCC (hexane–AcOEt
CH3); 13C-NMR (50 MHz, CD3OD): δ = 144.7 (s, C-triazole), 5 : 1) affording pure 11 (Rf = 0.19, 254 mg, 0.88 mmol, 60%
122.7 (d, C-triazole), 81.3 (d, C-1), 77.8 (d, C-2), 71.6 (d, C-3), yield) as a white solid.
66.7 (d, C-7a), 66.5 (t, CH3CH2O), 63.0 (t, OCH2triazole), 62.3
(t, C-8), 61.0 (d, C-6), 59.6 (t, C-5), 36.6 (t, C-7), 13.9 (q, CH3);
Benzylated tetravalent iminosugar 12
MS (ESI): m/z calcd for C13H22N4O4 + Na+ 321.15 [M + Na]+; To a solution of 4 (58 mg, 0.12 mmol) in 1.5 mL of a 2 : 1 THF–
found: 321.22; elemental analysis calcd (%) for C13H22N4O4 H2O mixture, CuSO4 (30 mol%, 2 mg, 0.009 mmol), sodium
(298.34): C 52.34, H 7.43, N 18.78; found: C 52.25, H 7.38, N ascorbate (60 mol%, 4 mg, 0.018 mmol) and 11 (10 mg,
18.72.
0.03 mmol) were added. The reaction mixture was heated in a
MW reactor at 80 °C for 45 min, until TLC analysis (PE–AcOEt
3 : 1) showed the disappearance of the starting material (Rf =
0.59) and formation of a new product (Rf = 0.00). After fil-
Benzylated trivalent iminosugar 10 from tripropargyl
amine (9)
To a solution of 4 (150 mg, 0.31 mmol) in 1.5 mL of a 2 : 1 tration through Celite®, the solvent was removed under
THF–H2O mixture, CuSO4 (30 mol%, 5 mg, 0.03 mmol), reduced pressure and the crude was purified by FCC (CH2Cl2–
sodium ascorbate (60 mol%, 12 mg, 0.06 mmol) and tripro- MeOH 15 : 1) affording pure 12 (Rf = 0.32, 61 mg, 0.023 mmol,
1
pargyl amine (9) (14 µL, 0.1 mmol) were added dropwise. The 91%) as a yellow oil. [α]2D2 = +3.43 (c = 0.35 in CHCl3); H-NMR
reaction mixture was heated in a MW reactor at 80 °C for (400 MHz, CDCl3): δ = 7.71 (s, 4H, H-triazole), 7.35–7.22 (m,
45 min, until TLC analysis (PE–AcOEt 3 : 1) showed the dis- 60H, H-Ar), 5.24–5.18 (m, 4H, H-6), 4.68–4.45 (m, 24H, H-Bn),
appearance of the starting material (Rf = 0.35) and formation 4.50 (s, 8H, OCH2triazole), 4.10–4.03 (m, 4H, H-2), 3.89 (t, J =
of a new product (Rf = 0.00). After filtration through Celite®, 4.9 Hz, 4H, H-1), 3.69 (td, J = 7.4, 4.8 Hz, 4H, H-7a), 3.58 (dd,
the solvent was removed under reduced pressure and the J = 9.3, 5.4 Hz, 4H, Ha-8), 3.54–3.50 (m, 4H, Hb-8), 3.50 (s, 8H,
crude was purified by FCC (CH2Cl2–MeOH 10 : 1 + 1% v/v of CCH2O), 3.36 (d, J = 5.3 Hz, 8H, H-5), 3.14–3.09 (m, 4H, H-3),
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2.42–2.35 (m, 4H, Ha-7), 2.21 (td, J = 13.6, 7.3 Hz, 4H, Hb-7); appearance of the starting material (Rf = 0.26) and formation
13C-NMR (50 MHz, CDCl3): δ = 145.2 (s, 4C, C-triazole), 138.4, of a new product (Rf = 0.92). After washing with a saturated
138.2, 137.9 (s, 12C, C-Ar), 128.4–127.6 (d, 60C, C-Ar), 121.6 (d, aqueous solution of NaHCO3 (3 × 25 mL) and H2O (25 mL),
4C, C-triazole), 88.4 (d, 4C, C-1), 85.8 (d, 4C, C-2), 73.2 (t, 4C, the combined organic layers were dried on Na2SO4, concen-
C-Bn), 72.4 (t, 4C, C-8), 72.3 (t, 4C, C-Bn), 71.9 (t, 4C, C-Bn), trated at reduced pressure and the crude mixture was purified
69.3, 69.0 (t, 4C, CCH2O), 68.9 (d, 4C, C-3), 66.7 (d, 4C, C-7a), by FCC (PE–AcOEt 5 : 1) affording pure 16 (Rf = 0.25, 614 mg,
65.1 (t, 4C, OCH2triazole), 60.9 (d, 4C, C-6), 60.2 (t, 4C, C-5), 2.07 mmol, 92% yield) as a colorless oil. 1H-NMR (400 MHz,
45.7 (s, CCH2O), 37.9 (t, 4C, C-7); IR (CDCl3): ν = 3088, 3066, CDCl3): δ = 7.78 (d, J = 8.3 Hz, 2H, H-Ar), 7.34 (d, J = 7.8 Hz,
3032, 2909, 2866, 1496, 1453, 1308, 1255, 1206, 1098 cm−1; MS 2H, H-Ar), 4.10 (d, J = 2.4 Hz, 2H, OCH2CuCH), 4.02 (t, J =
(ESI): m/z calcd for [(C133H148N16O16 + 2Na)/2]2+ 1135.57 6.3 Hz, 2H, H-1), 3.46 (t, J = 6.3 Hz, 2H, H-5), 2.45 (s, 3H, CH3),
[(M + 2Na)/2]+; found: 1136.10; elemental analysis calcd (%) 2.41 (t, J = 2.4 Hz, 1H, OCuCH), 1.71–1.64 (m, 2H, H-2),
for C133H148N16O16 (2226.70): C 71.74, H 6.70, N 10.06; found: 1.59–1.51 (m, 2H, H-4), 1.43–1.35 (m, 2H, H-3); 13C-NMR
C 71.26, H 6.27, N 9.85.
(50 MHz, CDCl3): δ = 144.7 (s, C-S), 133.1 (s, C-Ar), 129.8 (d,
2C, C-Ar), 127.8 (d, 2C, C-Ar), 79.9 (s, OCuCH), 74.2 (d,
OCHuCH), 70.4 (t, C-1), 69.6 (t, C-5), 58.0 (t, OCH2CuCH),
Polyhydroxylated tetravalent iminosugar 13
To a solution of 12 (96 mg, 0.043 mmol) in 10 mL of methanol, 28.7 (t, C-4), 28.5 (t, C-2), 22.0 (t, C-3), 21.6 (q, CH3); IR
48 mg of 10% Pd/C and two drops of 37% HCl were added (CHCl3): ν = 3306, 3031, 3019, 3010, 2957, 2867, 1598, 1495,
under a nitrogen atmosphere, and then the mixture was stirred 1455, 1358, 1188, 1175, 1097 cm−1; MS (ESI): m/z calcd for
under a hydrogen atmosphere at room temp. for 4 days. TLC C15H20O4S + Na+ 319.10 [M + Na]+; found: 319.05; elemental
analysis (CH2Cl2–MeOH 10 : 1) showed the disappearance of analysis calcd (%) for C15H20O4S (296.38): C 60.79, H 6.80,
the starting material (Rf = 0.57) and formation of a new S 10.82; found: C 60.67, H 6.72, S 10.69.
product (Rf = 0.00). The mixture was filtered through Celite®
Synthesis of trivalent scaffold 17 from pentaerythritol
and the solvent was removed under reduced pressure affording
a crude yellow oil. Then, the hydrochloride form was dissolved A solution of pentaerythritol (8 mg, 0.059 mmol) in 3 mL of
in MeOH and passed onto an ion exchange resin Dowex dry DMF was cooled to 0 °C and a solution of NaH (60% w/w
50WX8-200 eluting successively with MeOH, H2O and 6% in mineral oil, 60 mg, 1.44 mmol) and 16 (107 mg, 0.36 mmol)
NH4OH. Part of the compound was eluted in the first metha- in 3 mL of dry DMF was added dropwise. The reaction mixture
nolic fraction as hydrochloride salt, and a small part in the was stirred under a nitrogen atmosphere at room temp. for
final elution with ammonia as pure free amine 13 (6 mg, 20 h, until TLC analysis (PE–AcOEt 1 : 1) showed the disappear-
0.005 mmol, 12% yield). After treatment of the methanolic ance of the starting material (Rf = 0.00) and formation of a new
fraction with strongly basic resin Ambersep 900 OH pure 13 product (Rf = 0.73). The reaction was quenched with methanol
(38 mg, 0.033 mmol, 77% yield) was recovered as a waxy white and the solvent was removed under reduced pressure. After
solid (89% overall yield). [α]2D1 = +13.8 (c = 0.32 in MeOH); addition of AcOEt (15 mL), the combined organic layers were
1H-NMR (400 MHz, CD3OD): δ = 8.05 (s, 4H, H-triazole), 5.33 washed with H2O (2 × 15 mL), dried on Na2SO4 and concen-
(pquint, J = 5.7 Hz. 4H, H-6), 4.49 (s, 8H, OCH2triazole), 3.87 trated under reduced pressure. The crude mixture was purified
(t, J = 7.3 Hz, 4H, H-2), 3.82 (t, J = 7.3 Hz, 4H, H-1), 3.74 (dd, by FCC (PE–AcOEt 3 : 1) affording pure 17 (Rf = 0.18, 21 mg,
1
J = 11.2, 2.9 Hz, 4H, Ha-8), 3.59 (dd, J = 11.2, 5.3 Hz, 4H, Hb-8), 0.042 mmol, 71% yield) as a colorless oil. H-NMR (400 MHz,
3.49 (q, J = 6.8 Hz, 4H, H-7a), 3.43 (dd, J = 6.4, 2.0 Hz, 4H, Ha- CDCl3): δ = 4.12 (d, J = 2.4 Hz, 6H, OCH2CuCH), 3.68 (s, 2H,
5), 3.42 (s, 8H, CCH2O), 3.38 (dd, J = 5.9, 2.0 Hz, 4H, Hb-5), CH2OH), 3.50 (t, J = 6.6 Hz, 6H, CH2), 3.42 (s, 6H, CCH2O), 3.38
2.80 (ddd, J = 7.8, 5.8, 3.4 Hz, 4H, H-3), 2.49 (ddd, J = 13.2, 7.8, (t, J = 6.6 Hz, 6H, CH2), 2.42–2.40 (m, 3H, OCuCH), 1.64–1.53
5.6 Hz, 4H, Ha-7), 2.39 (dt, J = 13.2, 6.6 Hz, 4H, Hb-7); (m, 12H, CH2), 1.43–1.35 (m, 2H, CH2); 13C-NMR (50 MHz,
13C-NMR (50 MHz, CD3OD): δ = 143.8 (s, 4C, C-triazole), 121.8 CDCl3): δ = 80.0 (s, 3C, OCuCH), 74.1 (d, 3C, OCHuCH), 71.5
(d, 4C, C-triazole), 80.4 (d, 4C, C-1), 76.7 (d, 4C, C-2), 70.5 (d, (t, 3C, CCH2O), 71.5, 70.0 (t, 6C, CH2), 66.4 (t, CH2OH), 58.0 (t,
4C, C-3), 67.6 (t, 4C, CCH2O), 65.8 (s, CCH2O), 65.7 (d, 4C, 3C, OCH2CuCH), 44.7 (s, CCH2O), 29.3, 29.2 (t, 6C, CH2), 22.7
C-7a), 63.0 (t, 4C, OCH2triazole), 61.6 (t, 4C, C-8), 59.8 (d, 4C, (t, 3C, CH2); IR (CHCl3): ν = 3488, 3306, 3009, 2942, 2865,
C-6), 58.7 (t, 4C, C-5), 35.7 (t, 4C, C-7). MS (ESI): m/z calcd for 1459, 1358, 1094 cm−1; MS (ESI): m/z calcd for C29H48O7 + H +
C49H76N16O16 + Na+ 1167.55 [M + Na]+; found: 1167.78; Na+ 532.33 [M + H + Na]+; found: 532.58. Anal. Calcd for
elemental analysis calcd (%) for C49H76N16O16 (1145.23): C29H48O7 (508.69): C, 68.47; H, 9.51. Found C, 68.39; H, 9.41.
C 51.39, H 6.69, N 19.57; found: C 51.81, H 6.65, N 19.52.
Benzylated trivalent iminosugar 18
5-(Prop-2-in-1-yloxy)pentyl 4-methylbenzenesulfonate (16)
To a solution of 4 (78 mg, 0.16 mmol) in 3 mL of a 2 : 1 THF–
To a solution of 15 (320 mg, 2.25 mmol) in 30 mL of dry H2O mixture, CuSO4 (30 mol%, 2 mg, 0.015 mmol), sodium
CH2Cl2 were added dry NEt3 (1.57 mL, 11.25 mmol), DMAP ascorbate (60 mol%, 6 mg, 0.03 mmol) and 17 (27 mg,
(6.5 µL, 54 µmol) and tosyl chloride (1.29 g, 6.75 mmol). The 0.05 mmol) were added. The reaction mixture was heated in a
reaction mixture was stirred under a nitrogen atmosphere for MW reactor at 80 °C for 45 min, until TLC analysis (PE–AcOEt
15 h. Then, TLC analysis (AcOEt–PE 3 : 1) showed the dis- 3 : 1) showed the disappearance of the starting material (Rf =
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0.18) and formation of a new product (Rf = 0.00). After fil- 62.6 (t, CH2OAc), 62.4 (t, 3C, OCH2triazole), 60.4 (d, 3C, C-6),
tration through Celite®, the solvent was removed under 58.6 (t, 3C, C-5), 45.4 (s, CCH2O), 35.8 (t, 3C, C-7), 28.2 (t, 6C,
reduced pressure and the crude was purified by FCC (CH2Cl2– CH2), 21.7 (t, 3C, CH2), 18.5 (q, 10C, OAc); IR (CHCl3): ν =
MeOH 10 : 1) affording pure 18 (Rf = 0.26, 78 mg, 0.041 mmol, 3033, 3002, 2929, 2859, 1370, 1236, 1212 cm−1; MS (ESI): m/z
81%) as a waxy white solid. [α]D21 = +8.0 (c = 0.3 in CHCl3); calcd for C73H110N12O26 + Na+ 1593.75 [M + Na]+; found:
1H-NMR (400 MHz, CDCl3): δ = 7.63 (s, 3H, H-triazole), 1593.72; elemental analysis calcd (%) for C73H110N12O26
7.28–7.17 (m, 45H, H-Ar), 5.22–5.18 (m, 3H, H-6), 4.61–4.50 (1571.72): C 55.78, H 7.05, N 10.69; found: C 55.66, H 6.67, N
(AB system, J = 11.7 Hz, 6H, H-Bn), 4.51 (s, 6H, OCH2triazole), 10.35.
4.47–4.40 (AB system, J = 11.7 Hz, 6H, H-Bn), 4.46 (s, 6H,
H-Bn), 4.00 (t, J = 5.4 Hz, 3H, H-2), 3.83 (t, J = 4.6 Hz, 3H, H-1),
Polyhydroxylated trivalent iminosugar 20
3.61 (s, 2H, CH2OH), 3.55–3.43 (m, 6H, H-8), 3.46–3.43 (m, 3H, A suspension of 19 (36 mg, 0.024 mmol) and ion exchange
H-7a), 3.45 (t, J = 6.6 Hz, 6H, CH2), 3.34–3.28 (m, 6H, H-5), resin Ambersep 900 OH (600 mg) in 10 mL of methanol was
3.34 (s, 6H, CCH2O), 3.30 (t, J = 6.6 Hz, 6H, CH2), 3.10–3.06 (m, slowly stirred at room temp. for 16 h. After filtration of the
3H, H-3), 2.32–2.28 (m, 3H, Ha-7), 2.19–2.15 (m, 3H, Hb-7), resin on Celite®, the solvent was removed under reduced
1.58–1.44 (m, 12H, CH2), 1.35–1.26 (m, 6H, CH2); 13C-NMR pressure affording pure 20 (28 mg, 0.024 mmol, 100% yield)
(50 MHz, CDCl3): δ = 145.3 (s, 3C, C-triazole), 138.3, 138.0, as a yellow oil. [α]2D2 = +10.0 (c = 0.3 in MeOH); 1H-NMR
137.7 (s, 9C, C-Ar), 128.5–127.6 (d, 45C, C-Ar), 121.3 (d, 3C, (400 MHz, CD3OD): δ = 7.99 (s, 3H, H-triazole), 5.32–5.29 (m,
C-triazole), 88.3 (d, 3C, C-1), 85.8 (d, 3C, C-2), 73.3, 72.4 (t, 6C, 3H, H-6), 4.47 (s, 6H, OCH2triazole), 3.84–3.76 (m, 6H, H-1,
C-Bn), 72.3 (t, 3C, C-8), 72.0 (t, 3C, C-Bn), 71.6 (t, 6C, C-5, H-2), 3.69 (dd, J = 11.7, 3.4 Hz, 3H, Ha-8), 3.61–3.48 (m, 9H,
CH2), 70.8 (d or t, 6C, C-7a, CH2), 69.1 (d, 3C, C-3), 66.8 (t, Hb-8, H-7a, Ha-5), 3.46 (s, 2H, CH2OH), 3.46–3.38 (m, 3H, Hb-
CH2OH), 64.3 (t, 3C, OCH2triazole), 61.4 (d, 3C, C-6), 60.2 (t, 5), 3.42 (t, J = 6.6 Hz, 6H, CH2), 3.29–3.26 (m, 12H, CH2,
3C, CCH2O), 44.7 (s, CCH2O), 38.0 (t, 3C, C-7), 29.4 (t, 6C, CCH2O), 2.91–2.88 (m, 3H, H-3), 2.48 (ddd, J = 12.7, 7.6,
CH2), 22.7 (t, 3C, CH2); IR (CHCl3): ν = 3644, 3005, 2934, 2864, 5.1 Hz, 3H, Ha-7), 2.40 (dt, J = 12.7, 6.8 Hz, 3H, Hb-7),
1453, 1214, 1097 cm−1; MS (ESI): m/z calcd for C116H144N12O16 1.53–1.48 (m, 12H, CH2), 1.34–1.28 (m, 6H, CH2); 13C-NMR
+ Na+ 1984.07 [M + Na]+; found: 1984.88; elemental analysis (50 MHz, CD3OD): δ = 144.9 (s, 3C, C-triazole), 123.0 (d, 3C,
calcd (%) for C116H144N12O16 (1962.46): C 70.99, H 7.40, C-triazole), 80.5 (d, 3C, C-1), 77.5 (d, 3C, C-2), 72.5 (d, 3C, C-3),
N 8.56; found: C 71.0, H 7.68, N 8.45.
71.0, 70.2 (t, 6C, CH2), 69.5 (t, 3C, CCH2O), 68.0 (d, 3C, C-7a),
63.2 (t, 3C, OCH2triazole), 62.0 (t, CH2OH), 61.3 (t, 3C, C-8),
60.7 (d, 3C, C-6), 59.8 (t, 3C, C-5), 45.2 (s, CCH2O), 36.2 (t, 3C,
Peracetylated trivalent iminosugar 19
To a solution of 18 (67 mg, 0.035 mmol) in 15 mL of methanol, C-7), 29.0 (t, 6C, CH2), 22.6 (t, 3C, CH2); MS (ESI): m/z calcd
34 mg of 10% Pd/C and four drops of 37% HCl were added for C53H90N12O16 + Na+ 1173.65 [M + Na]+; found: 1173.59;
under a nitrogen atmosphere, then the mixture was stirred elemental analysis calcd (%) for C53H90N12O16 (1151.35):
under a hydrogen atmosphere at room temp. for 5 days. After C 55.29, H 7.88, N 14.60; found: C 55.11, H 7.65, N 14.32.
that TLC analysis (CH2Cl2–MeOH 10 : 1) showed the disappear-
Benzylated trivalent iminosugar 22
ance of the starting material (Rf = 0.26) and formation of a new
product (Rf = 0.00), the mixture was filtered through Celite® To a solution of 4 (141 mg, 0.291 mmol) in 3 mL of a 2 : 1
and the solvent was removed under reduced pressure. The THF–H2O mixture, CuSO4 (30 mol%, 4 mg, 0.026 mmol),
crude yellow oil was dissolved in pyridine (3 mL) and acetic sodium ascorbate (60 mol%, 10 mg, 0.052 mmol) and 21
anhydride (2 mL) was added. The solution was stirred at room (18 mg, 0.087 mmol) were added. The reaction mixture was
temp. for two days. After concentration under reduced heated in a MW reactor at 80 °C for 45 min, until TLC analysis
pressure, the crude mixture was purified by FCC (CH2Cl2– (PE–AcOEt 5 : 1) showed the disappearance of the starting
MeOH 30 : 1) affording pure 19 (Rf = 0.31, 40 mg, 0.027 mmol, material (Rf = 0.55) and formation of a new product (Rf = 0.00).
77%) as a pale yellow oil. [α]2D4 = +6.9 (c = 0.35 in CHCl3); After filtration on Celite®, the solvent was removed under
1H-NMR (400 MHz, CD3OD): δ = 7.99 (s, 3H, H-triazole), reduced pressure and the crude was purified by FCC (CH2Cl2–
5.41–5.35 (m, 3H, H-6), 5.34–5.31 (m, 3H, H-2), 5.10 (t, J = MeOH 12 : 1) affording pure 22 (Rf
= 0.37, 143 mg,
5.4 Hz, 3H, H-1), 4.55 (s, 6H, OCH2triazole), 4.21 (dd, J = 12.0, 0.086 mmol, 99%) as a yellow oil. [α]2D4 = +6.0 (c = 0.3 in
5.7 Hz, 3H, Ha-8), 4.08 (dd, J = 11.8, 5.9 Hz, 3H, Hb-8), 4.04 (d, CHCl3); 1H-NMR (400 MHz, CDCl3): δ = 7.76 (s, 3H, H-triazole),
J = 4.9 Hz, 2H, CH2OAc), 3.70 (td, J = 7.8, 5.4 Hz, 3H, H-7a), 7.34–7.23 (m, 45H, H-Ar), 5.30–5.20 (m, 3H, H-6), 4.76 (s, 2H,
3.52–3.35 (m, 12H, H-5, CCH2O), 3.50 (t, J = 6.6 Hz, 6H, CH2), OCH2triazole), 4.67–4.45 (m, 22H, H-Bn, OCH2triazole),
3.38–3.34 (m, 6H, CH2), 3.29–3.26 (m, 3H, H-3), 2.55–2.51 (m, 4.08–4.05 (m, 3H, H-2), 3.92–3.88 (m, 3H, H-1), 3.82 (pquint,
6H, H-7), 2.08–2.00 (m, 30H, OAc), 1.62–1.49 (m, 12H, CH2), J = 5.0 Hz, 1H, CH2CHCH2), 3.76–3.50 (m, 13H, H-7a, H-8,
1.43–1.37 (m, 6H, CH2); 13C-NMR (50 MHz, CD3OD): δ = 170.2 OCH2CHCH2O), 3.44–3.36 (m, 6H, H-5), 3.20–3.10 (m, 3H,
(s, CvO), 170.1 (s, 3C, CvO), 169.7 (s, 3C, CvO), 169.2 (s, 3C, H-3), 2.44–2.34 (m, 3H, Ha-7), 2.26–2.16 (m, 3H, Hb-7);
CvO), 143.9 (s, 3C, C-triazole), 121.5 (d, 3C, C-triazole), 79.8 13C-NMR (50 MHz, CDCl3): δ = 144.9 (s, 3C, C-triazole), 138.4,
(d, 3C, C-1), 73.4 (d, 3C, C-2), 70.0, 69.3, 68.1 (t, 9C, CH2, 138.1, 137.8 (s, 9C, C-Ar), 128.5–127.6 (d, 45C, C-Ar), 121.7 (d,
CCH2O), 66.0 (d, 3C, C-3), 65.7 (d, 3C, C-7a), 63.1 (t, 3C, C-8), 3C, C-triazole), 88.4 (d, 3C, C-1), 85.9 (d, 3C, C-2), 77.4 (d,
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CH2CHCH2), 73.3, 72.4, 72.0 (t, 11C, C-Bn, OCH2CHCH2O), 3.50 (pq, J = 6.8 Hz, 3H, H-7a), 3.42–3.36 (m, 6H, H-5), 2.80
70.4 (t, 3C, C-8), 69.1 (d, 3C, C-3), 66.8 (d, 3C, C-7a), 64.9, 64.8, (ddd, J = 8.4, 5.5, 3.1 Hz, 3H, H-3), 2.52–2.44 (m, 3H, Ha-7),
63.9 (t, OCH2triazole), 61.3 (d, 3C, C-6), 60.3 (t, 3C, C-5), 38.0 2.44–2.34 (m, 3H, Hb-7); 13C-NMR (50 MHz, CD3OD): δ = 145.9
(t, 3C, C-7); IR (CHCl3): ν = 3088, 3066, 3032, 2866, 1496, 1453, (s, 3C, C-triazole), 124.4 (d, 3C, C-triazole), 82.6 (d, 3C, C-1),
1365, 1249, 1207, 1111 cm−1. Elemental analysis calcd (%) for 79.0 (d, 3C, C-2), 78.9 (d, OCH2CHCH2O), 72.9 (d, 3C, C-3),
C99H110N12O12 (1660.01): C 71.63, H 6.68, N 10.13; found: C 71.2 (t, 2C, OCH2CHCH2O), 68.1 (d, 3C, C-7a), 65.2, 65.1, 64.2
71.51, H 6.60, N 10.08.
(t, OCH2triazole), 63.8 (t, 3C, C-8), 62.3 (d, 3C, C-6), 61.0 (t, 3C,
C-5), 38.0 (t, 3C, C-7); MS (ESI): m/z calcd for C36H56N12O12
Na+ 871.40 [M + Na]+; found: 871.50; elemental analysis calcd
+
Peracetylated trivalent iminosugar 23
To a solution of 22 (109 mg, 0.066 mmol) in 30 mL of metha- (%) for C36H56N12O12 (848.90): C 50.93, H 6.65, N 19.80; found:
nol, 60 mg of 10% Pd/C and four drops of 37% HCl were C 50.53, H 6.45, N 19.65.
added under a nitrogen atmosphere, then the mixture was
Synthesis of dendrimeric nonavalent scaffold 27
stirred under a hydrogen atmosphere at room temp. for 5 days.
TLC analysis (CH2Cl2–MeOH 10 : 1) showed the disappearance To a solution of 25 (75 mg, 0.32 mmol) and DIPEA (139 µL,
of the starting material (Rf = 0.67) and formation of a new 0.81 mmol) in dry CH2Cl2 (1 mL), cooled to 0 °C and under a
product (Rf = 0.00) The mixture was filtered through Celite® nitrogen atmosphere, a solution of 26 (24 mg, 0.09 mmol) in
and the solvent was removed under reduced pressure. The dry CH2Cl2 (1 mL) was added. The reaction mixture was stirred
crude yellow oil was dissolved in pyridine (3 mL), acetic anhy- at room temp. for 15 h, until TLC analysis (CH2Cl2–MeOH
dride (2 mL) was added and the reaction mixture was stirred at 10 : 1) showed the disappearance of the starting material (Rf =
room temp. for two days. Then, after concentration under 0.96) and formation of a new product (Rf = 0.84). After washing
reduced pressure, the crude mixture was purified by FCC with HCl 0.5 M (10 mL) and H2O (2 × 10 mL), the combined
(CH2Cl2–MeOH 10 : 1) affording pure 23 (Rf = 0.43, 44 mg, organic layers were dried over Na2SO4 and concentrated under
0.036 mmol, 54%) as a pale yellow oil. [α]2D2 = −2.3 (c = 0.94 in reduced pressure. The crude mixture was purified by FCC
CHCl3); 1H-NMR (400 MHz, CDCl3): δ = 7.79 (s, H-triazole), (hexane–AcOEt 2 : 1) affording pure 27 (Rf = 0.17, 53 mg,
7.73 (s, H-triazole), 7.72 (s, H-triazole), 5.32 (t, J = 5.3 Hz, 6H, 0.060 mmol, 68% yield) as a white solid.
H-6, H-2), 5.03 (t, J = 5.2 Hz, 3H, H-1), 4.74 (s, 2H, OCH2tri-
azole), 4.60 (s, 4H, OCH2triazole), 4.17–4.06 (m, 6H, H-8), 3.81
Benzylated nonavalent iminosugar 28
(pquint, J = 5.1 Hz, 1H, CH2CHCH2), 3.69–3.48 (m, 7H, H-7a, To a solution of 4 (218 mg, 0.45 mmol) in 2.4 mL of a 2 : 1
OCH2CHCH2O), 3.45–3.33 (m, 6H, H-5), 3.19 (pq, J = 5.9 Hz, THF–H2O mixture. CuSO4 (30 mol%, 2 mg, 0.014 mmol),
3H, H-3), 2.54–2.36 (m, 6H, H-7), 2.06–2.04 (s, 27H, OAc); sodium ascorbate (60 mol%, 6 mg, 0.029 mmol) and 27
13C-NMR (50 MHz, CDCl3): δ = 170.7, 170.4, 169.7 (s, 9C, (41 mg, 0.048 mmol) were added. The reaction mixture was
CvO), 145.4, 145.0, 144.9 (s, C-triazole), 121.8, 121.6, 121.5 (d, heated in a MW reactor at 80 °C for 45 min, until TLC analysis
C-triazole), 80.8 (d, 3C, C-1), 78.6 (d, 3C, C-2), 77.3 (d, (PE–AcOEt 3 : 1) showed the disappearance of the starting
CH2CHCH2), 70.2 (t, 2C, OCH2CHCH2O), 67.3 (d, 3C, C-3), 66.5 material (Rf = 0.35) and formation of a new product (Rf = 0.00).
(d, 3C, C-7a), 64.8 (t, 4C, C-8 and OCH2triazole), 64.6, 63.8 (t, After a filtration through Celite®, the solvent was removed
OCH2triazole), 61.1 (d, 3C, C-6), 60.0 (t, 3C, C-5), 37.4 (t, 3C, under reduced pressure and the crude was purified by FCC
C-7), 20.9 (q, 9C, CH3); IR (CHCl3): ν = 2942, 2867, 1740, 1626, (CH2Cl2–MeOH 20 : 1) affording pure 28 (Rf = 0.31, 214 mg,
1453, 1373, 1233, 1108, 1046 cm−1; MS (ESI): m/z calcd for 0.041 mmol, 91%) as a yellow oil. [α]2D2 = +3.21 (c = 0.53 in
C54H74N12O21 + Na+ 1249.50 [M + Na]+; found: 1249.50; CHCl3); 1H-NMR (400 MHz, CDCl3): δ = 8.34 (s, 9H, H-triazole),
elemental analysis calcd (%) for C54H74N12O21 (1660.01): C 7.69 (s, 3H, H-Ar), 7.32–7.19 (m, 135H, H-Ar), 5.19 (m, 9H,
52.85, H 6.08, N 13.70; found: C 52.87, H 5.94, N 13.72.
H-6), 4.66–4.55 (AB system, J = 12.2 Hz, 18H, Bn), 4.54–4.49 (m,
36H, H-Bn, OCH2triazole), 4.53–4.42 (AB system, J = 12.2 Hz,
18H, Bn), 4.05 (t, J = 5.4 Hz, 9H, H-2), 3.94 (s, 18H, CCH2O),
Polyhydroxylated trivalent iminosugar 24
A suspension of 23 (25 mg, 0.02 mmol) and ion exchange resin 3.89–3.86 (m, 9H, H-1), 3.69–3.66 (m, 9H, H-7a), 3.56 (dd, J =
Ambersep 900 OH (1000 mg) in 10 mL of methanol was slowly 9.3, 5.4 Hz, 9H, Ha-8), 3.50 (dd, J = 9.2, 6.4 Hz, 9H, Hb-8),
stirred at room temp. for 16 h. After filtration through Celite®, 3.34–3.32 (m, 18H, H-5), 3.11–3.08 (m, 9H, H-3), 2.39–2.34 (m,
the solvent was removed under reduced pressure and the 9H, Ha-7), 2.23–2.15 (m, 9H, Hb-7); 13C-NMR (50 MHz, CDCl3):
crude was purified through size exclusion chromatography by δ = 166.4 (s, 3C, CvO), 144.7 (s, 9C, C-triazole), 138.4, 138.2,
Sephadex G-10 (eluting with H2O) affording pure 24 (9 mg, 137.9 (s, 30C, C-Ar), 135.9 (s, 3C, Ar), 128.4–127.6 (d, 138C,
0.011 mmol, 55% yield) as a yellow oil. [α]2D5 = +20.8 (c = 0.48 C-Ar), 121.9 (d, 9C, C-triazole), 88.4 (d, 9C, C-1), 85.8 (d, 9C,
1
in MeOH); H-NMR (400 MHz, CD3OD): δ = 8.10–8.09 (m, 3H, C-2), 73.2 (t, 9C, C-Bn), 72.4 (t, 9C, C-Bn), 72.2 (t, 9C, C-Bn),
H-triazole), 5.33 (quint, J = 5.7 Hz, 3H, H-6), 4.73–4.71 (m, 2H, 71.9 (t, 9C, C-8), 68.9 (t or d, 18C, CCH2O, C-3), 66.8 (d, 9C,
OCH2triazole), 4.60–4.58 (m, 4H, OCH2triazole), 3.87 (t, J = C-7a), 64.8 (t, 9C, OCH2triazole), 61.0 (d, 9C, C-6), 60.8, (s, 3C,
7.4 Hz, 3H, H-2), 3.80 (t, J = 7.2 Hz, 3H, H-1), 3.78–3.75 (m, 1H, HNC-), 60.2 (t, 9C, C-5), 37.8 (t, 9C, C-7); IR (CHCl3): ν = 3088,
OCH2CHCH2O), 3.73 (dd, J = 11.3, 3.1 Hz, 3H, Ha-8), 3.64–3.54 3065, 3031, 2923, 2866, 1667, 1497, 1454, 1346, 1111,
(m, 4H, OCH2CHCH2O), 3.58 (dd, J = 11.3, 5.8 Hz, 3H, Hb-8), 1095 cm−1. Elemental analysis calcd (%) for C309H339N39O39
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(5223.24): C 71.05, H 6.54, N 10.46; found: C 70.54, H 6.15, 83.7 (d, C-6), 80.8 (d, C-1), 76.8 (d, C-2), 70.2 (d, C-3), 66.5 (d,
N 10.13.
C-7a), 62.8 (t, C-8), 59.4 (t, C-5), 37.0 (q, OMs), 36.2 (t, C-7); MS
(ESI): m/z calcd for C9H17NO6S + Na+ 290.07 [M + Na]+; found:
290.00; elemental analysis calcd (%) for C9H17NO6S (267.30):
C 40.44, H 6.41, N 5.24; found: C 40.31, H 6.87, N 4.75.
(1R,2R,3R,6R,7aR)-1,2-Bis(benzyloxy)-3-[(benzyloxy)methyl]-
6-(methylsulfonyloxy)-hexahydro-1H-pyrrolizine (29)
To a stirred solution of 3 (326 mg, 0.71 mmol) in dry CH2Cl2
(12 mL), NEt3 (298 μL, 2.13 mmol) was added under a nitrogen
atmosphere, and, at 0 °C, MsCl (71 μL, 0.92 mmol) was added
(1R,2R,3R,6S,7aR)-6-Azido-3-hydroxymethylhexahydro-1H-
pyrrolizidine-1,2-diol (31)
dropwise. The solution was left stirring at room temp. for 2 h. To a solution of 30 (108 mg, 0.40 mmol) in 4 mL of dry DMF
TLC analysis (AcOEt–PE 3 : 1) showed the disappearance of the sodium azide (52 mg, 0.80 mmol) was added. The reaction
starting material (Rf = 0.16) and formation of a new product mixture was stirred at 80 °C for 3 h, until TLC analysis
(Rf = 0.43). Then 20 mL of H2O was added, the mixture was (CH2Cl2–MeOH 3 : 1) showed the disappearance of the starting
extracted with CH2Cl2 and the organic layers were dried on material (Rf = 0.58) and formation of a new product (Rf = 0.50).
Na2SO4. Filtration through cotton and evaporation under Then, the solvent was removed under reduced pressure and
reduced pressure afforded crude 29 that was purified by FCC the crude was purified by FCC (CH2Cl2–MeOH 4 : 1) affording
(AcOEt–PE 1 : 1) affording pure 29 (Rf = 0.23, 317 mg, pure 31 (Rf = 0.38, 82 mg, 0.38 mmol, 95%) as a yellow oil.
1
0.59 mmol, 83% yield) as a colorless oil. [α]2D5 = −11.5 (c = 0.2 [α]D24 = +17.5 (c = 0.53 in MeOH); H-NMR (400 MHz, D2O): δ =
1
in CHCl3); H-NMR (400 MHz, CDCl3): δ = 7.37–7.25 (m, 15H, 4.31–4.27 (m, 1H, H-6), 3.73–3.66 (m, 2H, H-1, H-2), 3.63 (dd,
H-Ar), 5.20 (ddd, J = 8.3, 5.9, 2.9 Hz, 1H, H-6), 4.71–4.56 (AB J = 11.7, 3.4 Hz, 1H, Ha-8), 3.46 (dd, J = 11.7, 6.9 Hz, 1H, Hb-8),
system, J = 11.7 Hz, 2H, H-Bn), 4.59 (s, 2H, H-Bn), 4.57–4.50 3.17 (pq, J = 7.1 Hz, 1H, H-7a), 2.95 (dd, J = 11.7, 2.9 Hz, 1H,
(AB system, J = 11.7 Hz, 2H, Bn), 4.05–3.99 (m, 2H, H-1 e H-2), Ha-5), 2.80 (dd, J = 11.7, 4.9 Hz, 1H, Hb-5), 2.63 (ddd, J = 9.2,
3.59 (dd, J = 9.8, 3.9 Hz, 1H, Ha-8), 3.52 (dt, J = 8.3, 5.4 Hz, 1H, 7.1, 3.5 Hz, 1H, H-3), 2.02 (ddd, J = 13.1, 6.8, 3.4 Hz, 1H, Ha-7),
H-7a), 3.56 (dd, J = 9.8, 6.3 Hz, 1H, Hb-8), 3.33 (dt, J = 13.7, 1.88 (ddd, J = 13.1, 7.8, 5.3 Hz, 1H, Hb-7); 13C-NMR (50 MHz,
2.0 Hz, 1H, Ha-5), 3.29–3.25 (m, 1H, H-3), 3.21 (dd, J = 13.7, D2O) δ = 80.3 (d, C-1), 77.9 (d, C-2), 70.0 (d, C-3), 65.2 (d, C-7a),
4.9 Hz, 1H, Hb-5), 2.81 (s, 3H, Ms), 2.43 (ddd, J = 13.7, 7.8, 6.8 62.8 (d, C-6), 62.4 (t, C-8), 59.1 (t, C-5), 35.3 (t, C-7); MS (ESI):
Hz, 1H, Ha-7), 1.80 (dt, J = 13.7, 6.3 Hz, 1H, Hb-7); 13C-NMR m/z calcd for C8H14N4O3 + Na+ 237.10 [M + Na]+; found:
(50 MHz, CDCl3): δ = 138.1, 138.1, 138.0 (s, C-Ar), 128.4–127.8 237.08; elemental analysis calcd (%) for C8H14N4O3 (214.22): C
(d, 15C, C-Ar), 88.0 (d, C-1), 85.0 (d, C-2), 82.8 (d, C-6), 73.4 (t, 44.85, H 6.59, N 26.15; found: C 44.74, H 6.45, N 26.09.
C-Bn), 72.8 (t, C-Bn), 72.3 (t, C-Bn), 72.3 (t, C-8), 67.9 (d, C-3),
Polyhydroxylated nonavalent iminosugar 32
66.5 (d, C-7a), 59.5 (t, C-5), 38.7 (q, OMs), 37.8 (t, C-7); IR
(CHCl3): ν = 3088, 3053, 2986, 2831, 1951, 1808, 1606, 1422, To a solution of 31 (82 mg, 0.38 mmol) in 3 mL of a 2 : 1 THF–
1268, 1153; MS (ESI): m/z calcd for C30H35NO6S + H+ 538.22 H2O mixture, CuSO4 (30 mol%, 2 mg, 0.012 mmol), sodium
[M + H]+; found: 538.42; elemental analysis calcd (%) for ascorbate (60 mol%, 5 mg, 0.024 mmol) and 27 (34 mg,
C30H35NO6S (537.67): C 67.02, H 6.56, N 2.61; found: C 67.47, 0.04 mmol) were added. The reaction mixture was heated in a
H 6.87, N 2.31.
MW reactor at 100 °C for 2 h, until TLC analysis (EP–AcOEt
2 : 1) showed the disappearance of the starting material (Rf =
0.26) and formation of a new product (Rf = 0.00). After a
filtration through Celite®, the solvent was removed under
(1R,2R,3R,6R,7aR)-6-Methansulfonyloxy-3-
hydroxymethylhexahydro-1H-pyrrolizidine-1,2-diol (30)
To a solution of 29 (264 mg, 0.49 mmol) in 10 mL of methanol, reduced pressure and the crude was purified by FCC (MeOH–
132 mg of 10% Pd/C and two drops of 37% HCl were added CH2Cl2–NH4OH 2 : 1 : 1) affording pure 32 (Rf = 0.26, 62 mg,
under a nitrogen atmosphere, then the mixture was stirred 0.022 mmol, 55%) as a waxy white solid. [α]3D0 = +3.0 (c = 0.5 in
under a hydrogen atmosphere at room temp. for 16 h. 1H NMR H2O); 1H-NMR (400 MHz, D2O): δ = 7.87 (s, 9H, H-triazole),
control showed the disappearance of the starting material, 7.79 (s, 3H, H-Ar), 5.08–5.05 (m, 9H, H-6), 4.45 (s, 18H, OCH2-
then the mixture was filtered through Celite® and the solvent triazole), 3.76 (t, J = 7.4 Hz, 9H, H-1), 3.72–3.69 (m, 9H, H-2),
was removed under reduced pressure affording a crude yellow 3.70 (s, 18H, CCH2O), 3.62 (dd, J = 11.8, 3.4 Hz, 9H, Ha-8), 3.47
oil (168 mg). Free amine was obtained by passing the hydro- (dd, J = 11.8, 6.4 Hz, 9H, Hb-8), 3.33 (q, J = 6.8 Hz, 9H, H-7a),
chloride salt through a Dowex 50WX8 ion-exchange resin. 3.22 (dd, J = 12.2, 6.1 Hz, 9H, Ha-5), 3.13 (dd, J = 12.0, 5.8 Hz,
Elution with 6% NH4OH afforded the free base 30 (118 mg, 9H, Hb-5), 2.75–2.71 (m, 9H, H-3), 2.34–2.22 (m, 18H, H-7);
0.44 mmol, 90% yield) as a colourless oil. [α]2D9 = +19.0 (c = 13C-NMR (50 MHz, D2O): δ = 168.2 (s, 3C, CvO), 144.0 (s, 9C,
0.625 in MeOH); 1H-NMR (400 MHz, CD3OD): δ = 5.26–5.23 C-triazole), 135.3 (s, 3C, Ar), 129.1 (d, 3C, Ar), 124.0 (d, 9C,
(m, 1H, H-6), 3.95 (t, J = 8.0 Hz, 1H, H-1), 3.75 (dd, J = 11.2, 3.1 C-triazole), 80.1 (d, 9C, C-1), 76.9 (d, 9C, C-2), 70.1 (d, 9C, C-3),
Hz, 1H, Ha-8), 3.71 (dd, J = 9.0, 8.3 Hz, 1H, H-2), 3.55 (dd, J = 67.2 (t, 9C, CCH2O), 66.0 (d, 9C, C-7a), 63.5 (t, 9C, OCH2tri-
11.2, 5.9 Hz, 1H, Hb-8), 3.36 (dt, J = 13.9, 1.6 Hz, 1H, Ha-5), azole), 62.0 (t, 9C, C-8), 60.7 (s, 3C, CCH2O), 60.1 (d, 9C, C-6),
3.34 (dd, J = 8.0, 3.7 Hz, 1H, H-7a), 3.16 (dd, J = 13.9, 3.7 Hz, 59.0 (t, 9C, C-5), 35.6 (t, 9C, C-7); MS (ESI): m/z calcd for
1H, Hb-5), 3.12 (s, 3H, OMs), 3.02 (ddd, J = 9.1, 6.0, 3.1 Hz, 1H, [(C120H177N39O39 + 3Na)/3]3+ 952.42 [(M + 3Na)/3]+; found:
H-3), 2.35–2.23 (m, 2H, H-7); 13C-NMR (50 MHz, CD3OD): δ = 953.17; elemental analysis calcd (%) for
C120H177N39O39
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(2789.93): C 51.66, H 6.39, N 19.59; found: C 51.59, H 6.33,
N 19.42.
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The experiments were performed essentially as previously
described.41 The percentage of inhibition towards the corres-
ponding glycosidase was determined in the presence of 1 mM
of the inhibitor on the well. Each enzymatic assay (final
volume 0.12 mL) contained 0.01 to 0.5 units mL−1 of the
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visible absorption spectroscopy at 405 nm. Under these con-
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linear with both reaction time and concentration of the
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of enzyme activity) and Ki towards the corresponding glyco-
sidase were calculated. IC50 values were calculated from plots
of percentage of inhibition versus inhibitor concentration. The
Ki values were determined from the Lineweaver–Burk plots
(see ESI†). Each experiment (%, IC50 and Ki) was performed in
duplicate and the average values were given.
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Acknowledgements
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We thank MIUR-Italy (PRIN 2008, 200824M2HX, and PRIN
2010-2011, 2010L9SH3 K 006) and Ente Cassa di Risparmio di
Firenze (CRF, 2009/3141) for financial support. The research
leading to these results has received funding from the Euro-
pean Research Council under the European Union’s Seventh
Framework Programme (FP7/2007-2013)/ERC grant agreement
no. [291349] on photonic micro robotics. We thank Ministerio
de Economia
y
Competitividad (CTQ2012-31247) of
Spain, Junta de Andalucía (FQM 345) and European Commu-
nity FP7 program (HEALTH-F2-2011-256986-project acronym
PANACREAS) for financial support. We also thank Mr Simone
Pisano for preliminary experiments.
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