Design and synthesis of new amino-modified iminocyclitols: Selective inhibitors of α-galactosidase

Article abstract of DOI:10.1039/b926123k

A new and short synthesis of hitherto unreported stereo analogue of amino-modified five-membered iminocyclitols from readily available tri-O-benzyl-d-glucal is described. Significantly, glycosidase inhibition studies of alkylamino substituted iminocyclitols display a high degree of selectivity towards α-galactosidase.

Full text of DOI:10.1039/b926123k

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COMMUNICATION
www.rsc.org/obc | Organic & Biomolecular Chemistry
Design and synthesis of new amino-modified iminocyclitols: selective inhibitors
of a-galactosidase†‡§
Muthupandian Ganesan, Rekhawar V. Madhukarrao and Namakkal G. Ramesh*
Received 11th December 2009, Accepted 2nd February 2010
First published as an Advance Article on the web 15th February 2010
DOI: 10.1039/b926123k
A new and short synthesis of hitherto unreported stereo
analogue of amino-modified five-membered iminocyclitols
from readily available tri-O-benzyl-D-glucal is described.
Significantly, glycosidase inhibition studies of alkylamino
substituted iminocyclitols display a high degree of selectivity
towards a-galactosidase.
aglycon moieties of iminocyclitols, in order to fine tune their
inhibitory activities against glycosidases, is becoming an area
of increasing importance. Pioneering work on such functional
group modifications on five-membered iminocyclitols was car-
ried out by Wong et al. who had observed that a synthetic
azasugar 6 obtained by replacement of one of the side chain
hydroxyl groups of naturally occurring 2,5-dihydroxymethyl-3,4-
dihydroxy pyrrolidine (DMDP) 4 by an acetamido group had
pronounced inhibitory activity against N-acetylglucosaminidase.4
Subsequently, a high throughput screening of a library of such
1-aminodeoxy-DMDP (ADMDP) analogues, synthesized by
them, resulted in the identification of novel structures for antivirals
and osteoarthritis.5 Stu¨tz and coworkers have carried out extensive
research on the synthesis of C-1 amino-modified five-memebered
iminocyclitols such as 5 by meticulously exploiting the Amadori
rearrangement of 5-azido-5-deoxy-D-glucofuranose and similar
compounds with dibenzylamine as a key step and investigated,
in detail, their structure–activity relationship against various
glycosidases.6 Since the significant findings that such synthetically
designed azasugars are promising structures for various disorders,
there has been an upsurge in research related to their synthesis
and biological studies. A recent report on the first structural
basis of glycosidase inhibition by five-membered azasugars has
further spurred the research activities in this area.2a Besides
the glycosidase inhibition activities, transition metal complexes
of amino-substituted polyhydroxypyrrolidines have been used as
catalysts for asymmetric transformations and find applications in
medicines as well.7
Over the years, the chemistry and biology of naturally occurring
iminocyclitols (azasugars) and their synthetic analogues have
received immense research attention due to their significant
and selective inhibition of various glycosidases,1 a feature at-
tributable to their structural resemblance as well as mimicry of
the glycosidase oxocarbenium-ion transition state.2 The successful
launch of synthetically modified six-membered azasugar based
R
clinical drugs such as Glysetꢀ 2 (for non-insulin dependant
R
diabetes) and Zavescaꢀ 3 (for the control of Gaucher’s disease)
(Fig. 1) has provided an added impetus for research in this
area.3 As a consequence, functional group modifications at the
A few other strategies so far reported for the synthesis of
ADMDP 5 and its various stereoisomers include (i) synthetic
manipulations of naturally occurring DMDP 4 and its stereo
analogues by way of selective conversion of one of the side chain
hydroxyl groups into an amino functionality;5b,8 (ii) intramolecular
cyclization of a C2-symmetric amino alcohol;9 (iii) nucleophilic
ring opening of sugar based bis-aziridines;10 (iv) transformation
of an optically active g-lactam in to ADMDP analogues;11
and (v) nucleophilic addition of cyanide to sugar based cyclic
nitrones followed by reduction.7a,12 Postel and co-workers very
recently disclosed the synthesis of 2-aminocyclopropyl polyhy-
droxylated pyrrolidine and its isomers through cyclopropanation
of glycoaminonitriles.13 To the best of our knowledge, there is no
glycal based approach available for the synthesis of such amino-
modified azasugars, despite their ready availability. Earlier we had
reported a novel one-pot diamination of glycals with chloramine-
T and utilized them for efficient syntheses of N-linked glycoamino
acids and peptides.14 We expected that the pluripotent diamine 8
could as well serve as a versatile precursor for the synthesis of
amino-modified azasugar 10, through a cleavage-recombination
Fig. 1 Representative examples of azasugars.
Department of Chemistry, Indian Institute of Technology-Delhi, Hauz Khas,
New Delhi, 110016, India. E-mail: ramesh@chemistry.iitd.ac.in; Fax: +91-
11-26581102; Tel: +91-11-26596584
† Dedicated to Prof. Yasuyuki Kita on the occasion of his 65th birthday.
‡ Part of this work was presented as a poster at the 15th European
Carbohydrate Symposium held at University of Vienna, Vienna, Austria,
19–24 July 2009.
§ Electronic supplementary information (ESI) available: Experimental
procedure, spectral data, and copies of 1H-NMR and 13C-NMR spectra of
all compounds; detailed procedure for enzyme assay studies including
the source of enzymes, methodology, range of activities. See DOI:
10.1039/b926123k
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pathway. In this communication, we report a new and a very short
synthesis of a hitherto unreported stereo analogue of ADMDP
from readily available tri-O-benzyl-D-glucal 7 via cleavage of
the O–C1 bond of diamine 8 followed by an intramolecular
cyclization of the diamino alcohol intermediate 9. The glycosidase
inhibition studies of these molecules reveal that azasugars with
an alkylamino-substituted side chain display a high degree of
selectivity towards a-galactosidase (from green coffee bean).
Our synthesis of amino-modified iminocyclitols started with
the reduction of the readily available diamine 814 to the diamino
alcohol 9, which was easily accomplished in 95% yield by refluxing
8 with 1.5 equiv. of LiAlH4 for 20 min (Scheme 1). It was expected
that an intramolecular cyclization of compound 9 could readily
provide the pyrrolidine derivative 10. Thus, when compound 9
was initially subjected to an intramolecular cyclization under
Mitsunobu conditions, complete consumption of starting material
was noticed (TLC) in 30 min and formation of a new product
was observed. After purification and thorough characterization,
the product was confirmed to be the polyhydroxypyrrolidine 10
and not the piperidine derivative 11 (Scheme 1). In the 1H-NMR
spectrum of the product, the signal of the NH proton (that
disappeared upon D2O exchange) resonated as a triplet with a
coupling constant of 7.2 Hz, thereby indicating that there were
two neighbouring protons for the NH to couple with. This clearly
established the structure of the product to be the pyrrolidine 10.
Both the neighbouring diastereotopic methylene protons were
also found to couple with the NH proton as revealed by the
1H–1H COSY spectrum of the above compound. On the other
hand, in the case of piperidine derivative 11, the signal of the
NH proton was expected to resonate only as a doublet due to
its coupling with the lone neighbouring CH proton. Irrespective
of the reaction conditions (change in temperature, solvent, time
etc.) and other reagents {(PPh3, I2, imidazole); (NaH, MsCl,
THF)} employed, compound 10 was always obtained as the
exclusive product in the intramolecular reaction of 9. Among
them, the best yield (90%) was obtained when the reaction
was carried out under Mitsunobu conditions at 0 ◦C to room
temperature with 1.3 equiv. of PPh3 and 1.4 equiv. of DEAD
in THF as a solvent. In order to study the inhibition properties
it was imperative to deprotect the tosyl and benzyl groups of
10 to obtain the parent polyhydroxypyrrolidine 12. This was
conveniently achieved in a single step by treating compound
10 with Na/liq.NH3 to obtain (2S,3R,4R,5S)-2-aminomethyl-5-
hydroxymethyl-3,4-dihydroxypyrrolidine 12 in 86% yield. Thus, we
have accomplished a very short synthesis of a new stereo-analogue
of ADMDP in just three steps and with a total yield of 74% from
8 (Scheme 1).
We next focused our attention towards the synthesis of a
few derivatives of 12, possessing different substituents at the
side chain amino functionality, with a view to investigating the
role of substituents on their inhibition activities. An obvious
choice was the synthesis of the tosyl derivative 15, for which
selective deprotection of the tertiary tosyl group of 10 in the
presence of the side chain secondary one was an essential requisite.
After several experiments, we were successful in chemoselectively
deprotecting the tertiary tosyl group of 10 over the secondary one
by treating it with Na–Hg. Compound 14 was obtained in 96%
yield. Subsequent catalytic hydrogenation of 14 in presence of 10%
Pd/C gave the tosylamido polyhydroxypyrrolidine 15 in 90% yield
(Scheme 2).
Scheme 2 Reagents and conditions: (a) Ac2O, DMAP, pyridine, rt, 24 h,
95%; (b) 3% Na–Hg, Na2HPO4, DMF–MeOH (4 : 0.5), 60 ◦C, 3 h; (c) 10%
Pd/C, H2(g), MeOH, 30 ◦C, 5 h, 90%.
While a library of compounds bearing diverse functional groups
on the side chain nitrogen of ADMDP have been reported in
the literature,4–13 those possessing simple alkyl groups were visibly
missing and thus their glycosidase inhibition studies as well as
structure–activity relationship largely remain uninvestigated. This
prompted us to explore the possibility of synthesizing a few alkyl
derivatives of 12 and undertake their inhibitory studies against
glycosidases. Thus, reaction of 10 with alkyl halides in presence
of sodium hydride afforded the alkyl-substituted derivatives 16
and 17 in good yields. Unexpectedly, global deprotection on
compounds 16 and 17 using Na/liq. NH3 did not give the required
products 20 and 21. Instead, cleavage of the alkyl groups also
occurred along with the other protecting groups resulting in
the formation of ADMDP 12, which was rather unusual and
Scheme 1 Reagents and conditions: (a) LiAlH4, THF, reflux, 20 min, 95%;
(b) Ph3P, DEAD, THF, 0 ◦C to rt, 30 min, 90%; (c) Na–liq.NH3, THF,
-78 ◦C, 3 h, 86%.
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Table 1 Glycosidase inhibition studies
surprising. However, this problem was circumvented by following
an alternate two-step procedure. On treatment with Na–Hg,
compounds 16 and 17 underwent a smooth didetosylation reaction
to give products 18 and 19 in high yields. Hydrogenation of
compounds 18 and 19 in presence of 10% Pd/C and HCl delivered
the required products 20 and 21 respectively (Scheme 3).
IC50, mM
12 15
Enzyme (source)
20
21
22
a-glucosidase type I (baker’s yeast)
b-glucosidase (almond)
a-galactosidase (green coffee beans)
b-galactosidase (Escherichia coli)
n.i. 3.5
n.i. 6.3
n.i. n.i.
n.i. 5.4
n.i.
n.i.
6.9
n.i.
n.i.
n.i.
8.1
n.i.
n.i.
n.i.
n.i.
n.i.
n.i., no inhibition was observed up to 12 mM inhibitor concentration
not inhibit a-galactosidase. Quite contrastingly, both the alkyl
derivatives 20 and 21 displayed a very high degree of selectivity by
inhibiting only a-galactosidase among the four glycosidases tested.
Thus, the complementary behaviour of 15 versus 20 and 21 toward
various enzymes is not only interesting but also could provide
vital information for further understanding the structural basis
of glycosidase inhibition by these amino-modified five-membered
iminocyclitols.
In conclusion, we have developed a glycal based route towards
the synthesis of a new stereo analogue of amino-modified poly-
hydroxypyrrolidines. This methodology is one of the shortest
(only 3 to 5 steps from the diamine 8) routes available for
the synthesis of ADMDP analogues. The glycosidase inhibitory
activities of these compounds are quite interesting. Notably, the
specific inhibitory nature of alkyl-ADMDP compounds 20 and 21
towards a-galactosidase is expected to spur further research on the
synthesis and biological studies of alkylamino derivatives, which
has largely remained elusive so far. Detailed investigation on the
nature of the interaction between these inhibitors and the enzymes
is currently underway. Further, since inhibitors of a-galactosidase
have been found to be promising chemical chaperones for Fabry’s
disease,15 compounds 20 and 21 reported here could find potential
applications in this direction as well.
Scheme 3 Reagents and conditions: (a) NaH, RX, DMF, rt; (b) 3%
Na–Hg, Na2HPO4, DMF–MeOH (4 : 0.5), 60 ◦C, 3 h; (c) (i) 10% Pd/C,
H2(g), dil. HCl, MeOH, 30 ◦C, 12 h, (ii) Et3N.
Our next target was the synthesis of the acetyl derivative 22.
Acetylation of compound 10 using standard procedures afforded
the N-acetyl-N-tosyl derivative 13 in 95% yield (Scheme 2), which
however, on treatment with Na–Hg did not undergo the expected
didetosylation reaction. Unlike compounds 16 and 18, which
underwent smooth didetosylation reaction with Na–Hg, in the
case of 13, it was the side chain acetyl and not the tosyl group that
was cleaved along with the tosyl group attached to the ring nitrogen
to afford compound 14 exclusively (Scheme 2). Such a preference
of acetyl over tosyl group cleavage of N-acetyl-N-tosylamides with
Na–Hg has hardly been reported in literature and thus could prove
to be a useful protocol in organic synthesis. Finally, synthesis
of the acetamido polyhydroxypyrrolidine 22 was carried out by
direct acetylation of the free amine 12 with acetic anhydride under
solvent-free conditions using a modified procedure developed in
our lab (Scheme 4).
Acknowledgements
The authors thank the Department of Science and Technology,
India for financial support and Dr (Ms) Ruchi Gaur for her help
in the enzyme assay studies.
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