New leads for selective inhibitors of α-L-fucosidases. Synthesis and glycosidase inhibitory activities of [(2R,3S,4R)-3,4-dihydroxypyrrolidin-2-yl]furan derivatives

Article abstract of DOI:10.1016/S0960-894X(01)00497-8

Readily derived from D-glucose, 5-[(2R,3S,4R)-3,4-dihydroxypyrrolidin-2-yl]-2-methyl-3-furoic esters and amides are selective and competitive inhibitors (K_i ≥ 3 μM) of α-L-fucosidase from bovine epididymis and from human placenta.

Full text of DOI:10.1016/S0960-894X(01)00497-8

Bioorganic & Medicinal ChemistryLetters 11 (2001) 2555–2559
New Leads for Selective Inhibitors of ꢀ-L-Fucosidases.
Synthesis and Glycosidase Inhibitory Activities of
[(2R,3S,4R)-3,4-Dihydroxypyrrolidin-2-yl]furan Derivatives
Inmaculada Robina,a,* Antonio J. Moreno-Vargas,a Jose G. Fernandez-Bolanos,a
Jose Fuentes,a Raynald Demangeb and Pierre Vogelb,*
a
´
Departamento de Quımica Organica, Facultad de Quımica, Universidad de Sevilla, E-41071 Sevilla, Spain
´
´
Section de Chimie de l’Universite de Lausanne, BCH, CH-1015 Lausanne-Dorigny, Switzerland
b
´
Received 19 March 2001; accepted 13 July2001
Abstract—Readilyderived from d-glucose, 5-[(2R,3S,4R)-3,4-dihydroxypyrrolidin-2-yl]-2-methyl-3-furoic esters and amides are
selective and competitive inhibitors (Kiꢀ3 mM) of a-l-fucosidase from bovine epididymis and from human placenta. # 2001
Elsevier Science Ltd. All rights reserved.
Inhibitors of glycosidases are useful tools for the study
of the biological function of oligosaccharides.1 Theyare
also potential drugs against diseases where the control
of oligosaccharide metabolism can be linked to cellular
dysfunction.2 The control of N-linked oligosaccharides
biosynthesis to alter cell surface expression in malignant
cell3 or affect viral membrane protein folding and
assembly,4 has therapeutic implications.5 l-Fucose resi-
due in sialyl Lewis X tetrasaccharide expressed on the
surface of leukocyte and some tumor cells is essential
for their adhesion to the endothelial-leukocyte adhesion
molecules.6 Fucosidase in invasive human ovarian car-
cinoma cell mediates degradation of the subendothelial
extracellular matrix.7a Inhibitors of a-l-fucosidases
inhibit the cytopathic effect of HIV and reduces infec-
tion.7b,8 These findings have stimulated the invention of
different a-l-fucosidase inhibitors, the most potent, at
the moment, 1,5-dideoxy-1,5-imino-l-fucitol (1),9 exhi-
bits Ki values of 3–10 nM. Such remarkable inhibition
constants reflect some of the strongest interactions of
carbohydrate analogues with proteins known to date.
Monosaccharide mimics often lack protein specificity;
furthermore, to become a drug a good inhibitor must
ization at one of the chiral centers, N-alkylation12 or
introduction of a-aminomethyl13 or other alkyl
14
groups12,13 reduces the inhibitorypower significantly.
Even l-fucosamidrazone 2 is less active than 1 byat
least a factor of 100.15 More successful is the imine 3
which is almost as active as 1.16 Removal of the methyl
group of 1 produces the moderate inhibitors 412 and 517
confirming that 1 does not allow much changes to be
made to its structure without losing its inhibitoryactiv-
itysignificantly. This fact has stimulated the exploration
of other structures such as iso-fuco-fagomine 612 which
is not better than 4 except if it bears a gem-diamine
moietyas in 7 and derivatives with the exocyclic amine
N-acylated.18 The latter compounds probablylack the
necessarychemical stabilityfor potential drug develop-
ment. More stable fucose mimics is the 5a-carba-a-l-
fucopyranosylamine 8, 200 times less active than 1.
When N-conjugated with anhydroaldoses, no better
activitywas observed. 19 Azepines 9, 1020 and guanidino-
sugars 11, 1221 have been reported. The results suggest
that, contraryto 1, N-alkylation can improve the inhi-
bitoryactivityof these systems. The thiosugar 13 has a
moderate inhibitoryactivitythat can be enhanced upon
conjugation (glycosidation) with a lypophilic phenyl
system such as 1422 or a b-d-galactoside (15).23,24 Ami-
nocyclopentitol 16 that imitates the transition structure
of the hydrolysis of an a-d-fucopyranoside is a good
inhibitor of a-l-fucosidase.25 Its potencyand selectivity
can be improved through N-benzylation (see 17).26
10
satisfya number of conditions such as stabilityin the
stomach and membrane permeability, which often
requires the presence of lipophilic moieties that simple
sugar mimetics do not have. Anymodification of 1 such
as methyl side chain extension or removal,11,12 epimer-
A competitive and specific a-l-fucosidase inhibitor
could be an l-fucopyranosyl cation mimic to which one
*Corresponding authors. Fax: +34-95-462-4960; e-mail: robina@cica.es
(I. Robina); pierre.vogel@ico.unil.ch (P. Vogel)
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00497-8

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I. Robina et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2555–2559
Table 1. Examples of a-l-fucosidase inhibitors (Ki optimal pH)a,b,c
attaches substituents able to recognize the rim of the
enzyme active site through its shape (bulk that repels
water molecules), specific electrostatic (e.g., H-bonds)
or/and lypophilic interactions. Since the piperidine ana-
logues of l-fucose do not permit wide modifications
without losing inhibitoryactivity(the exception being
perhaps 316) we envisioned to use a more flexible cis-3,4-
dihydroxypyrrolidine base27 and to attach to it ade-
quate groups.28 The data reported for 18–23 in Table 1
do not contradict this plan.12,27,30 We disclose pre-
liminaryresults showing that this concept applies for
the inhibition of a-l-fucosidases by5-[(2 R,3S,4R)-3,4-
dihydroxypyrrolidin-2-yl]-furan derivatives 24–28.
and ethyl acetylacetate.31 Selective tosylation of its pri-
maryalcohol (TsCl/pryidine,
ꢁ15 ꢂC, 2 h) provided
tosylate 30 in 57% yield. Treatment of 30 with N-
chlorosuccinimide and dimethylsulfide32 (CH2Cl2,
ꢁ20 ꢂC) gave a mixture of chlorides that reacted with
benzylamine to give a unique pyrrolidine 31 in 48%
yield. After debenzylation (H2/Pd–C, EtOH) pure 24a
was obtained in 97% yield. N-Protection with CbzCl
and acetylation gave 32 in quantitative yield. Its struc-
ture was established byits spectral data and confirmed
byNOE experiments when comparing with its epimer
on C-2033 that showed a NOE between proton pairs H-
20/H-30 that is not observed in compound 32. Reduction
of 24a with LiAlH4 in THF furnished alcohol 25 (90%).
Saponification of 32 gave the furoic acid 33 that was
converted into a small libraryof esters 24 (R=Me, i-
Pr), amides 27 (R=Et, i-Pr, Ph) and 28 after hydro-
genolysis. Direct esterification and amidification of 26
(obtained bysaponification of 24a) led to polymer
formation. The N-protected derivative 33 was used to
prepare esters 24 (+ROH activation with 2,6-dichloro-
benzoyl chloride, pyridine in DMF), amides 27
(+RNH2), 28 (Et2NH; activation with PyBOP/DIPEA
The synthesis of 24–28 starts from the known furan
derivative obtained in one single step from d-glucose
Scheme 1.

I. Robina et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2555–2559
2557
in DMF). Deprotection of the pyrrolidine moiety was
done byhdyrogenolsyis (10% Pd/charcoal, MeOH)
(Scheme 1).
the following enzymes: a-galactosidases from Aspergil-
lus niger and from E. coli; b-galactosisades from E. coli,
from Aspergillus orizae; maltase from yeast, from rice;
isolmaltase from baker yeast; b-glucosidase from
almonds, from Caldocellum saccharolyticum; b-manno-
sidase from Helix pomatia, a-N-acetylgalactosaminidase
from chicken liver; b-N-acetylglucosaminidase from
jack bean, from bovine epididymis A and B. As shown
in Table 2, esters 24 as well as amides 27a,c and 28 are
good inhibitors of a-l-fucosidases from bovine epididy-
mis and human placenta. The inhibitoryactivityis
We have tested compounds 24a,b,c, 25, 26, 27a,c,d and
28 for their inhibitoryactivities toward 25 commercially
available glycosidases. The data are summarized in
Table 2 for two a-l-fucosidases, one a-l-galactosidase,
three b-d-galactosidases, two amyloglucosidases and
two a-mannosidases. These compounds did not show
anyinhibitoryactivityat 1 mM concentration toward
Table 2. Inhibitoryactivities of pyrrolidine derivatives. Percentage of inhibition at 1 mM, IC 50 and Ki in mM, when measured. Optimal pH, 35 ꢂCa,b
Enzyme/Compound
24a
24b
24c
25
26
27a
27c
17d
28
a-l-Fucosidase
Bovine epididymis
84%
IC50=200
Ki=9
86%
IC50=100
Ki=6.5
91%
IC50=50
Ki=4.9
NI
NI
91%
IC50=40
Ki=3.2
94%
IC50=40
Ki3.0
<10%
85%
IC50=110
Ki=9.1
Human placenta
76%
IC50=300
Ki=15
88%
IC50=150
Ki=13.8
92%
IC50=160
Ki=8.6
NI
NI
92%
IC50=80
Ki=4.8
93%
IC50=80
Ki=5.3
51%
IC50=1000
86%
IC50=220
Ki=20.1
a-Galactosidase
Coffee bean
NI
42%
29%
NI
NI
NI
NI
NI
NI
b-Galactosidase
Bovine liver
57%
IC50=850
54%
IC50=800
57%
IC50=640
50%
30%
52%
38%
39%
58%
Aspergillus niger
80%
IC50=370
Ki=340c
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
Jack bean
Amyloglucosidase
Aspergillus niger
42%
NI
NI
NI
NI
NI
ND
NI
23%
33%
27%
44%
NI
NI
NI
NI
NI
23%
33%
ND
ND
NI
Rhizopus mold
a-Mannosidase
Jack bean
25%
26%
25%
38%
23%
78%
IC50=250
Ki=85
NI
NI
29%
32%
NI
NI
27%
31%
Almonds
NI
36%
57%
NI
NI
29%
NI
NI: no inhibition at 1 mM; ND: not determined.
aFor the conditions of measurements see ref 34.
bThe mode of inhibition for which Ki given is competitive.
cMixed type of inhibition.
Figure 1. Effect of p-nitrophenyl a-fucopyranoside (S) concentration on a-l-fucosidase inhibition byvarious concentrations of 27a. The data were
plotted according to the Lineweaver–Burk method. (A) bovine epididymis, (B) human placenta.

2558
I. Robina et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2555–2559
much weaker for the phenyl amide 27d, suggesting size
restriction for the groups that can be attached to the
carboxylic moiety of these systems. Interestingly, both
the alcohol 25 and the corresponding carboxylic acid 26
are inactive. For compounds showing more than 50%
inhibition at 1 mM, IC50 and/or Ki values have been
measured.34 Except for inhibition of b-galactosidase
from Aspergillus niger byester 24a that showed a mixed
type of inhibition, all our new a-l-fucosidases inhibitors
are competitive (see Fig. 1) with p-nitrophenyl a-l-
fucopyranoside, thus confirming that they recognize the
active site of the enzymes as designed initially. Some of
our a-l-fucosidase inhibitors inhibit other glycosidases
weakly(Table 2). Nevertheless it appears that amides
27a and 27c that are our most potent inhibitors are also
the most selective.
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We thank the Direccion General de Investigacion Cien-
tıfica yTe cnica of Spain (Grant PB 97-730), the Junta
de Andalucıa (FQM 134) and the Swiss National
Science Foundation for financial support. We also
thank the Universityof Seville and Socrates-Erasmus
(Lausanne/Seville) for a fellowship to A.J.M.-V. This
work is part of the European COST D13/0001/99.
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