Synthesis and evaluation of aminocyclopentitol inhibitors of β-glucosidases

Article abstract of DOI:10.1021/ol991252b

(matrix presented) (1R,2S,3S,4R,5R)-4-Amino-5-(hydroxymethyl)cyclopentane-1,2,3-triol 1, prepared from D-glucose, inhibits β-glucosidases from Caldocellum saccharolyticum (K_i = 1.8 x 10^-7 M) and from almonds (K_i = 3.4 x 10

Full text of DOI:10.1021/ol991252b

ORGANIC
LETTERS
2000
Vol. 2, No. 2
151-154
Synthesis and Evaluation of
Aminocyclopentitol Inhibitors of
â-Glucosidases
Olivier Boss, Emmanuel Leroy, Adrian Blaser, and Jean-Louis Reymond*
Department of Chemistry and Biochemistry, UniVersity of Bern,
Freiestrasse 3, 3012 Bern, Switzerland
jean-louis.reymond@ioc.unibe.ch
Received November 17, 1999
ABSTRACT
(1R,2S,3S,4R,5R)-4-Amino-5-(hydroxymethyl)cyclopentane-1,2,3-triol 1, prepared from D-glucose, inhibits â-glucosidases from Caldocellum
saccharolyticum (K ) 1.8 × 10-7 M) and from almonds (K ) 3.4 × 10-6 M). Inhibition is not influenced by N-ethylation (f 15) but is strongly
i
i
reduced upon N-acetylation (f 12). Inversion of stereochemistry at C(5) (f 14) has little effect on inhibition of â-glucosidases. These experiments
suggest that 1 acts as an analogue of a protonated â-glucoside.
Glycosidase inhibitors can be used for treating diabetes,
cancer, and viral (HIV, influenza) and bacterial infections
and as insecticides. Most of these inhibitors are mono-
saccharide analogues displaying a basic nitrogen-containing
function near the anomeric center, such as deoxynojirimycin
and isofagomine.1 Aminocyclopentitols such as mannostatin
(5) are also powerful inhibitors of glycosidases.2 However,
the relationship between inhibitor structure and inhibition
remains poorly understood in this series. We have shown
recently that aminocyclopentitols designed as mimics of R-
or â-configured protonated glycosides are potent anomer-
selective inhibitors of glycosidases.3 Herein we report an
efficient synthesis of aminocyclopentitol 1 as a mimic of a
protonated â-glucoside and show that this compound is a
potent inhibitor of â-glucosidases. Comparison of its inhibi-
tion pattern with close analogues provides the first evidence
that these inhibitors indeed act as cationic mimics of the
protonated glycosides.
The reaction mechanism of glycosidic bond cleavage is
acid catalyzed and involves protonation of the exocyclic
oxygen atom at the anomeric center to give a protonated
glycoside 2 (Scheme 1).4 Subsequent rate-limiting cleavage
Scheme 1. Mechanism of Acid-Catalyzed Glycosidic Bond
Cleavage for the Example of a â-Glucoside
(1) Recent reviews: (a) Bols, M. Acc. Chem. Res. 1998, 31, 1. (b)
Heightman, T. D.; Vasella, A. T. Angew. Chem. 1999, 111, 795. (c) Davies,
G.; Sinnott, M. L.; Withers, S. G. In ComprehensiVe Biological Catalysis;
Sinnott, M., Ed.; Academic Press: London, 1998; pp 119-208.
(2) Berecibar, A.; Grandjean, C.; Siriwardena, A. Chem. ReV. 1999, 99,
779.
of the C(1)-O(1) bond leads to oxocarbonium ion interme-
diate 3, which then reacts with a molecule of water to form
(3) Leroy, E.; Reymond, J.-L. Org. Lett. 1999, 1, 775.
(4) Bennet, A. J.; Sinnott, M. L. J. Am. Chem. Soc. 1986, 108, 7287.
10.1021/ol991252b CCC: $19.00 © 2000 American Chemical Society
Published on Web 12/23/1999

the product. The true transition state of the reaction lies in
between 2 and 3, which are high-energy intermediates. The
enzymatic mechanism is similar, although 2 and 3 may not
occur as intermediates due to a transition from specific acid
catalysis (pre-equilibrium protonation) to general acid ca-
talysis (rate-limiting protonation) and the formation of a
covalent intermediate with some enzymes.5 Despite the
differences between solution and enzymatic reaction mech-
anisms, analogues of the high-energy intermediates along
the chemical reaction pathway are generally potent glycosi-
dase inhibitors.
polyspecificity of type I â-glucosidases, prompted us to
investigate the synthesis and evaluation of the parent â-gluco-
configured aminocyclopentitol 1. Functionalized aminocy-
clopentitols, including natural products such as mannostatin,
can be prepared by a variety of synthetic routes.2,8 One of
the first and most straightforward approaches, reported by
Bartlett et al. in 1988,9 involves the radical cyclization of
oxime 8 to form benzylated hydroxylamines 9a and 9b.
Oxime 8 is itself easily obtained in two steps from tetra-O-
benzylglucose (Scheme 3). The strategy of closing the
Glycosidases are generally highly specific in cleaving only
one type of glycoside in a given anomeric configuration.
Most glycosidase inhibitors have been designed as analogues
of the oxocarbonium ion intermediate 3, where the stereo-
chemistry of the substrate’s anomeric configuration is lost.
Recently we proposed that anomer-selective glycosidase
inhibitors could be rationally designed by mimicking instead
the protonated glycoside 2 using aminocyclopentitols. In
particular, the amino substituent, in a well-defined R or â
configuration, can be used as a mimic of the protonated
oxygen atom O(1) and thus encodes for the anomeric
specificity of the inhibitor. Thus, aminocyclopentitol 4
(Scheme 2), an analogue of a protonated R-mannoside related
Scheme 3. Synthesis of Aminocyclopentitol 1 and Related
Structuresa
Scheme 2. Structure of Aminocyclopentitol Glycosidase
Inhibitors
a Reagents and conditions: (a) (i) NH2OBn, MeOH, pyridine,
90 °C, 3.5 h (84%), (ii) ClCSOPh, CH2Cl2, pyridine, 0°C, 3.5 h
(73%); (b) Bu3SnH, AIBN, benzene, reflux, 4 h (39% 9a, 27 %
9b); (c) Zn, AcOH, 135 °C, 4 h (32% 10a + 43 % 11a, 19% 10b
+ 28 % 11b); (d) 10 Atm H2, Pd/C, EtOH, 25 °C, 10 days (100%);
(e) HCl 1.2 N, reflux, 15 h (100%); (f) HCl 1.2 N, AcOH, H2,
Pd/C (100%).
to mannostatin 5 and reported by Farr et al.,6 is a potent
inhibitor of R-mannosidase (IC50 ) 62 nM for jack beans
R-mannosidase). While compound 6, an analogue of a
protonated â-L-fucoside, is only a weak inhibitor for R-L-
fucosidase (Ki ) 28 µM for bovine kidney R-L-fucosidase),7
the related aminocyclopentitol 7, which mimics a protonated
â-galactoside, inhibits â-galactosidases (Ki ) 3 µM) and
â-glucosidases (Ki ) 0.25 µM) selectively over R-galactosi-
dase (Ki ) 25 µM) and R-glucosidase (IC50 ) 100 µM).3
The potent inhibition of the galacto-configured inhibitor
7 with â-glucosidases, which can be explained by the known
cyclopentane ring by radical cyclization between C(1) and
C(5) in an hexose oxime, which has been extended subse-
quently by a number of groups,10 allows one to incorporate
the stereochemical pattern of the carbohydrate into the target
(8) (a) Aoyagi, T.; Yamamoto, T.; Kojiri, K.; Morishima, H.; Nagai,
M.; Hamada, M.; Takeuchi, T.; Umezawa, H. J. Antibiot. 1989, 42, 883.
(b) King, S. B.; Ganem, B. J. Org. Chem. 1994, 59, 562. (c) Maloisel,
J.-L.; Vasella, A.; Trost, B. M.; van Vranken, D. L. HelV. Chim. Acta 1992,
75, 1515. (d) Blattner, R.; Furneaux, R. H.; Kemmitt, T.; Tyler, P. C.;
Ferrier, R. J.; Tide´n, A.-K. J. Chem. Soc., Perkin Trans. 1 1994, 3411. (e)
Blattner, R.; Furneaux, R. H.; Lynch, G. P. Carbohydr. Res. 1996, 294,
29. (f) Li, J.; Lang, F.; Ganem, B. J. Org. Chem. 1998, 63, 3403. (g) Uchida,
C.; Kimura, H.; Ogawa, S. Bioorg. Med. Chem. 1997, 5, 921.
(9) Bartlett, P. A.; McLaren, K. L.; Ting, P. C. J. Am. Chem. Soc. 1988,
110, 1633.
(5) The C(1)-O(1) bond cleavage precedes the protonation step in the
enzymatic hydrolysis with phenolic leaving groups. Protonation does not
occur with acidic leaving groups such as pyridine or fluoride. Review on
mechanisms: Sinnott, M. L. Chem. ReV. 1990, 90, 1171-1202.
(6) Farr, R. A.; Peet, N. P.; Kand, M. S. Tetrahedron Lett. 1990, 31,
7109.
(7) Blaser, A.; Reymond, J.-L. HelV. Chim. Acta 1999, 82, 760.
152
Org. Lett., Vol. 2, No. 2, 2000

Table 1. Inhibition Data on Glycosidasesa
Ki(1)
× 10-6
Ki(12)
enzyme
origin
M
× 10-6
M
â-glucosidase
â-glucosidase
R-glucosidase
â-galactosidase
â-galactosidase
R-galactosidase
â-mannosidase
R-mannosidase
C.s.b
almond
yeast
E. coli
bovine liver
green coffee beans
snail acetone powder
jack beans
0.18
3.4
13
170
5.4
(28%)
(23%)
0.60
6.4
19
(9%)
59
(11%)
(6%)
9.1
0.16
2.6
12
(9%)
37
(20%)
(9%)
115
41
(11%)
(23%)
(16%)
(19%)
(7%)
(16%)
(19%)
180
a 100 µL assays contained the indicated enzyme at 0.1 U/mL, and the corresponding nitrophenyl glycosides in 0.1 M HEPES buffer at pH 6.8, at 25 °C.
The reactions were followed in individual wells of flat-bottom, 96-well, half-area polystyrene cell culture plates (Costar) using a UV Spectramax 250
instrument from Molecular Devices. The competitive inhibition constants Ki are given in micromoles and were determined by a Dixon plot of inhibition. No
data are given when less than 5% inhibition was observed. For weak inhibition, the percentage of inhibition observed with [S] ) 1 mM, [I] ) 0.1 mM is
b
given in parentheses. C.s. ) Caldocellum saccharolyticum. The following substrate and inhibitor concentrations were used. â-Glucosidase C.s.: [S] ) 1.0
and 0.3 mM; [1] ) 0, 62.5 nM, 125, 250, 500, 1000 nM; [12] ) 0, 12.5, 25, 50, 100, 200, 400 µM; [13] ) 0, 1, 2, 5, 10, 20, 50 µM; [14] ) 0, 0.3125, 0.625,
1.25, 2.5, 5 µM; [15] ) 0, 0.0625, 0.125, 0.25, 0.5, 1, 2 µM. Almond â-glucosidase: [S] ) 1.0 and 0.3 mM; [1] ) 0, 0.2, 0.5, 1, 2, 5,10 µM; [14] ) 0, 2,
5, 10, 20, 50, 100 µM; [15] ) 0, 0.3125, 0.625, 1.25, 2.5, 5, 10 µM. Yeast R-glucosidase: [S] ) 0.3 and 0.1 mM; [1] ) [14] ) 0, 3.125, 6.25, 12.5, 25,
50, 100 µM; [15] ) 0, 12.5, 25, 50, 100, 200 µM. Bovine liver â-galactosidase: [S] ) 0.9 and 0.3 mM; [1] ) [14] ) [15] ) 0, 6.25, 12.5, 25, 50, 100, 200
µM. Jack beans R-mannosidase: [S] ) 0.78 and 0.26 mM; [1] ) [15] ) 0, 6.25, 12.5, 25, 50, 100, 200 µM; [14] ) 0, 6.25, 12.5, 25, 50, 100 µM. Enzymes
and substrates were purchased from Fluka or Sigma.
aminocyclopentitol and highlights the structural analogies
between the two compounds.
inhibitor, 1 mM substrate) were characterized in more detail
by measurement of the competitive inhibition constant.
The results show that 1 is a potent inhibitor of â-glucosi-
dases. It is comparable in potency with the â-galacto-
configured stereoisomer 7 and clearly belongs to the more
potent small molecular weight glycosidase inhibitors. The
isomeric aminocyclopentitol 14, which derives from depro-
tection of the minor isomer formed during the radical
cyclization and corresponds to an R-L-ido configuration, also
turns out to be a potent inhibitor of â-glucosidases. As noted
for the â-galacto-configured aminocyclopentitol 7, this cross-
reactivity is explained by the known polyspecificity of type
I â-glucosidases.1,11 This should not hide the fact that the
relative configuration of aminocyclopentitols influences
inhibition of the different glycosidases in a manner consistent
with their design as analogues of protonated glycosides. Thus,
the â-gluco-configured inhibitor 1 displays the most pro-
nounced selectivity for â-glucosidases, while its â-galacto-
configured isomer 7 is the only one in the series to strongly
inhibit â-galactosidases. By contrast the R-L-ido-configured
inhibitor 14 displays an unexpected cross-reactivity with
R-mannosidase. Finally all of these inhibitors show a good
â-anomer selectivity in agreement with their amino substitu-
ent being on the â-face.
Protected hydroxylamines 9a/b were obtained by following
the literature procedure9 and were easily separated by column
chromatography. The complete debenzylation of 9a necessary
to prepare 1, which had not been investigated by Bartlett et
al., proved unexpectedly challenging. After experimenting
with a variety of schemes, deprotection succeeded by first
cleaving the N-O bond in 9a with zinc in acetic acid to
give 10a and the corresponding amide 11a. While direct
hydrogenation of amine 10a only gave mixtures of products,
extensive hydrogenation of amide 11a proceeded cleanly and
quantitatively to give 12. Finally, acidic hydrolysis of 12
with 1.2 N HCl gave 1 as the only product as the
hydrochloride salt. A similar sequence starting with isomer
9b gave sequentially 10b and 11b, the amide 13, and finally
aminocyclopentitol 14. Reduction of amide 12 with hydrogen
and palladium in acidic medium gave the N-ethyl derivative
15. The structure and stereochemistry of the compounds was
confirmed by analytical data. In particular, NOE in the
amides 12 and 13 allowed for the establishment of the
relative configuration of the amino and hydroxymethyl
substituents and confirmed the original structural assignment
for 9a and 9b.9
Aminocyclopentitol 1 was assayed for inhibition of gly-
cosidases together with its stereoisomer 14, the corresponding
acetamides 12 and 13, and its N-ethyl derivative 15 (Table
1). All measurements were carried out with the corresponding
nitrophenyl glycoside substrates in aqueous buffer at pH 6.8,
25 °C, under which conditions all enzymes displayed
satisfactory activity. All enzyme-inhibitor pairs displaying
more than 30% inhibition in the initial screening (100 µM
N-Acetylation to form the neutral amides 12 and 13
essentially abolishes inhibition in both 1 and 14. By contrast,
the N-ethyl derivative 15 shows an inhibition pattern almost
identical with that of 1. The loss of inhibition potency in 12
or 13 is therefore probably not due to a steric effect of the
added acetyl group but truly reflects the importance of the
basic amino group, which is protonated and bears a positive
charge at neutral pH, for inhibition by aminocyclopentitols
1 and 15. On this basis one can reasonably propose that these
inhibitors are binding to the enzymes as analogues of the
(10) a) Simpkins, N. S.; Stokes, S.; Whittle, A. J. J. Chem. Soc., Perkin
Trans. 1 1992, 2471. (b) Takahashi, S.; Inoue, H.; Kuzuhara, H. J.
Carbohydr. Chem. 1995, 14, 273. (c) Boiron, A.; Zillig, P.; Faber, D.; Giese,
B. J. Org. Chem. 1998, 63, 5877. (d) Storch de Gracia, I.; Dietrich, H.;
Bobo, S.; Chiara, J. L. J. Org. Chem. 1998, 63, 5883.
(11) (a) Henrissat, B. Biochem. J. 1991, 280, 309. (b) Bauer, M. W.;
Bylina, E. J.; Swanson, R. V.; Kelly, R. M. J. Biol. Chem. 1996, 271, 23749.
Org. Lett., Vol. 2, No. 2, 2000
153

protonated â-glucoside 2 (Scheme 1). This interpretation is
further supported by the inhibition selectivities discussed
above. The key interaction explaining the potent inhibition
observed is probably a salt bridge between the enzyme’s
carboxyl group that protonates the substrate’s leaving group
and the inhibitor’s ammonium group. A similar interaction
explains that glycosylamines are also glycosidase inhibitors.12
In conclusion, we have shown that the â-gluco-configured
aminocyclopentitol 1 is a potent and selective inhibitor of
â-glucosidases. The presence of a basic amino group and
the relative configuration of the substituents were shown to
be essential for both inhibition potency and selectivity. In
particular, the essential role of the positively charged amino
group was demonstrated by abolishing inhibition via acety-
lation. The deprotection sequence reported here from the
benzyl-protected hydroxylamine intermediates to the free
aminocyclopentitols should be applicable for the rapid
synthesis of related aminocyclopentitols.
Acknowledgment. This work was supported by the
University of Bern, the Swiss National Science Foundation,
and the Wander Stiftung.
(12) Ki(â-D-galacto-pyrannosylamine) ) 7 µM for Escherichia coli
â-galactosidase (in this case an enzyme-bound Mg2+ activates the leaving
group): Legler, G.; Herrchen, M. Carbohydr. Res. 1983, 116, 95. Ki(R-D-
manno-pyrannosylamine) ) 300 µM for jack beans R-mannosidase: Maity,
S. K.; Dutta, S. K.; Banerjee, A. K.; Achari, B.; Singh, M. Tetrahedron
1994, 50, 6965. Ki(L-fuco-pyrannosylamine) ) 1.2 µM for bovine kidney
R-L-fucosidase: Legler, G.; Stu¨tz, A. E.; Immich, H. Carbohydr. Res. 1995,
272, 17. Ki(â-D-gluco-pyrannosylamine) ) 24 µM for â-glucosidase from
Trichoderma reesei: Kolarova, N.; Trgina, R.; Linek, K.; Farkas, V.
Carbohydr. Res. 1995, 273, 109.
Supporting Information Available: Data for compounds
9a, 9b, 12, 1‚HCl, 13, 14‚HCl, and 15‚HCl. Dixon plot of
inhibition with 1 and â-glucosidases from C. saccharolyticum
and from almonds. This material is available free of charge
via the Internet at http://pubs.acs.org.
OL991252B
154
Org. Lett., Vol. 2, No. 2, 2000