α-L-fucosidase inhibition by pyrrolidine-ferrocene hybrids: Rationalization of ligand-binding properties by structural studies

Article abstract of DOI:10.1002/chem.201301001

Enhanced metabolism of fucose through fucosidase overexpression is a signature of some cancer types, thus suggesting that fucosidase-targetted ligands could play the role of drug-delivery vectors. Herein, we describe the synthesis of a new series of pyrrolidine-ferrocene conjugates, consisting of a L-fuco-configured dihydroxypyrrolidine as the fucosidase ligand armed with a cytotoxic ferrocenylamine moeity. Three-dimensional structures of several of these fucosidase inhibitors reveal transition-state-mimicking ³E conformations. Elaboration with the ferrocenyl moiety results in sub-micromolar inhibitors of both bovine and bacterial fucosidases, with the 3D structure of the latter revealing electron density indicative of highly mobile alkylferrocene compounds. The best compounds show a strong antiproliferative effect, with up to 100 % inhibition of the proliferation of MDA-MB-231 cancer cells at 50 μM. Transition-state-mimicking ³E conformations (see picture) are evident from the three-dimensional structures of ferrocenyl iminosugar/ fucosidase complexes. Novel pyrrolidine-ferrocene conjugates show strong anti-fucosidase and antiproliferative action, with up to 100 % inhibition of proliferation of an MDA-MB-231 cancer-cell line at 50 μM. Copyright

Full text of DOI:10.1002/chem.201301001

FULL PAPER
DOI: 10.1002/chem.201301001
a-l-Fucosidase Inhibition by Pyrrolidine–Ferrocene Hybrids:
Rationalization of Ligand-Binding Properties by Structural Studies
Audrey Hottin,[a] Daniel W. Wright,[b] Agata Steenackers,[c] Philippe Delannoy,[c]
Faustine Dubar,[d, e] Christophe Biot,[c] Gideon J. Davies,[b] and Jean-Bernard Behr*[a]
Abstract: Enhanced metabolism of
fucose through fucosidase overexpres-
sion is a signature of some cancer
types, thus suggesting that fucosidase-
targetted ligands could play the role of
drug-delivery vectors. Herein, we de-
scribe the synthesis of a new series of
pyrrolidine–ferrocene conjugates, con-
sisting of a l-fuco-configured dihydrox-
ypyrrolidine as the fucosidase ligand
armed with a cytotoxic ferrocenyla-
mine moeity. Three-dimensional struc-
tures of several of these fucosidase in-
hibitors reveal transition-state-mimick-
the ferrocenyl moiety results in sub-mi-
cromolar inhibitors of both bovine and
bacterial fucosidases, with the 3D
structure of the latter revealing elec-
tron density indicative of highly mobile
alkylferrocene compounds. The best
compounds show a strong antiprolifera-
tive effect, with up to 100% inhibition
of the proliferation of MDA-MB-231
cancer cells at 50 mm.
3
ing E conformations. Elaboration with
Keywords: antitumor agents · gly-
cosidases · hydrolases · inhibition ·
protein structures
Introduction
have received much attention due to the central role of fu-
cosylated conjugates. Indeed, many antigenic oligosacchar-
ides are functionalized by a terminal a-l-fucose moiety, thus
suggesting that this 6-deoxyhexose acts as a physiopathologi-
cal effector.[3] The biosynthesis and degradation of fucosides
in vivo is performed by FucT and AFU, respectively, but the
regulatory mechanisms for fucosylation are not fully under-
stood. Nevertheless, elevated levels of both fucosyltransfer-
ases and fucosidases have been detected in various cancer
tissues and the action of FucT and AFU during metastatic
events is supported by several lines of evidence.[4] As a con-
sequence of this overexpression, fucosidase is a potential re-
ceptor for selective targeting of cancer tissues. Ligands that
display high affinity for fucosidases (or fucosyltransferases)
should have utility as drug-delivery vectors for the detection
or control of malignancy.[5] Herein, we focus on five-mem-
bered iminocyclitols as potent fucosidase ligands modified
The glycosylation of proteins located at the cell surface
drives many biological events, such as cell–cell interactions,
cell migration, bacterial adhesion, and viral invasion.[1] It
has also been shown that perturbations of host glycans con-
tributes to tumour invasion and metastasis.[2] An aberrant
glycosylation pattern is associated with differential expres-
sion of the glycosidase and glycosyltransferase enzymes re-
quired for the biosynthesis of oligosaccharides. Among
these, a-l-fucosidases (AFU) and fucosyltransferases (FucT)
[a] A. Hottin, Dr. J.-B. Behr
Universitꢀ de Reims Champagne-Ardenne
Institut de Chimie Molꢀculaire de Reims, CNRS UMR 7312
UFR des Sciences Exactes et Naturelles
51687 Reims Cedex 2 (France)
Fax : (+33)326913166
with a cytotoxic ferrocenylACHTNUTRGNEaNUG mine moiety.
E-mail: jb.behr@univ-reims.fr
Iminosugars are natural or synthetic sugar mimics in
which the ring oxygen atom has been replaced by a nitrogen
atom. At physiological pH value, the positively charged am-
monium centre of imino and aza sugars (i.e., those in which
a ring carbon atom has been replaced by a nitrogen atom)
contributes to mimicry of the oxocarbenium ion-like transi-
tion state of the enzyme-catalyzed reaction and, facilitated
by charge–charge interactions, gives rise to strong affinities
(recently reviewed by Gloster and Davies[6a]). Indeed,
whereas the dissociation constant KM of a glycosidase–glyco-
side Michaelis complex ranges from 10 to 1000 mm, iminosu-
gars show 3–6 orders of magnitude more potent affinities
with Ki values in the micro- to nanomolar range.[6] Hence,
iminosugars disrupt the normal processing of glycosidases
(and to a lesser extent some glycosyltransferases); a proper-
[b] D. W. Wright, Prof. G. J. Davies
Structural Biology Laboratory
Department of Chemistry
University of York, York YO105DD (UK)
[c] A. Steenackers, Prof. P. Delannoy, Prof. C. Biot
Universitꢀ Lille Nord de France, Universitꢀ Lille 1
Unitꢀ de Glycobiologie Structurale et Fonctionnelle
CNRS UMR 8576, IFR 147
59650 Villeneuve dꢁAscq Cedex (France)
[d] Dr. F. Dubar
Universitꢀ Lille Nord de France, Universitꢀ Lille 1
Unitꢀ de Catalyse et Chimie du Solide, CNRS UMR 8181
59652 Villeneuve dꢁAscq Cedex (France)
[e] Dr. F. Dubar
Present address: School of Chemistry
University of Glasgow, Glasgow, G128QQ (UK)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201301001.
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ty that has been exploited for the development of new ther-
apeutic agents.[7]
A number of fucosidase-inhibiting imino and aza sugars
have been studied (Figure 1). Piperidines, exemplified by 1
and 2, feature a specific fucopyranose-like stereochemical
agents. The crystal structures of some five-membered imino-
cyclitols
complexed
with
BtFuc2970
reveal
a
3E conformation for inhibitor binding, thus mimicking the
3
postulated H4 conformation of the catalytic transition state
and, where present, clearly show the ferrocenyl moiety
pointing toward the solvent molecules. The new compounds
show sub-micromolar inhibition of fucosidase and antiproli-
ferative action with up to 100% inhibition of MDA-MB-231
cancer cells proliferation at 50 mm.
Results and Discussion
Synthesis of fucosidase inhibitors: The synthesis of the
target ferrocenyl–pyrrolidines 5a–c was envisioned by cou-
pling the cytotoxic ferrocenylamine moiety with a formyl–
pyrrolidine species through reductive amination (Figure 2).
Figure 1. Structures of fucosidase inhibitors.
configuration and have been shown to be tightly binding in-
hibitors of fucosidase from various origins.[8] Intriguingly,
five-membered iminocyclitols, such as 3 and 4, show compa-
rable performances, although they lack one hydroxy func-
tion in their structures.[9] Furthermore, the presence of aryl
substituents in the pseudoanomeric position of either the pi-
peridine or pyrrolidine framework improves the inhibition
potencies, likely through hydrophobic effects in the aglycon
binding pocket.[10] In contrast with the piperidine ana-
logues,[11] so far little is known about the structural bases for
AFU inhibition by five-membered iminocyclitols. Such in-
formation would aid the design of new potent ligands as
therapeutic candidates.
We have previously reported ferrocene–iminocyclitol hy-
brids 4a–c as the models of such drug-carrier conjugates.[12]
In anticancer therapy, ferrocene (Fc) derivatives have been
shown to generate a strong cytotoxic effect, which was relat-
ed to the production of reactive oxygen species (ROS)
through ferrocene-initiated Fenton reactions.[13] Structurally,
compounds 4a–c result from the combination of the cytotox-
ic ferrocenylamine moiety as the drug and an l-fuco-config-
ured dihydroxypyrrolidine as the carrier. The resulting hy-
brids harness both the AFU binding properties and the cyto-
toxicity of each moiety alone. Among these compounds, 4c
was the most active of the series displaying IC50 =1.2 mm
toward fucosidase and 77% growth inhibition of MDA-MB-
231 breast-cancer cells at 50 mm. In continuation of our pre-
vious work, structure–activity relationship studies were ex-
tended to new analogues to uncover the mode of binding of
these innovative organometallic inhibitors.
Figure 2. Synthetic access to new ferrocenyl iminosugars 5a–c.
Whereas the required amines 6a–c of general structure Fc-
AHCTNUGTERNG(NNU CH2)nNH2 (n=1, 2, and 3 for 6a–c, respectively) may be
obtained by following known synthetic procedures,[12] a mul-
tistep synthesis is required for the preparation of an enantio-
merically pure formyl–pyrrolidine moiety such as A. A large
panel of methods has been reported to access C2-functional-
ized iminocyclitols.[14] Among these methods, the addition of
organometallic compounds to glycosylamines is particularly
well suited and has been used with success for the synthesis
of formyl–pyrrolidine derivatives related to other sugar
series.[15] Thus, by using the ribose-derived N-benzylglycosyl-
amine 7 as the starting material, 2-vinylpyrrolidine 8 was ob-
tained in two steps after the stereoselective addition of vi-
nylmagnesium bromide and subsequent treatment with
methanesulfonyl chloride (Scheme 1). This method resulted
in the selective formation of the targeted (2S)-2-vinylpyrroli-
dine 8, which displays the required configuration for strong
binding with AFU. Unfortunately, all our attempts to trans-
form the double bond into a formyl group failed.
The direct ozonolysis of a tertiary amine such as 8 must
be performed on the corresponding sulfate or hydrochloride
salts to avoid oxidation at the nitrogen atom.[15a,c] In our
case, the treatment of acid-sensitive 8 with either HCl or
H2SO4 might lead to deprotection of the acetonide group.
For this reason, ozonolysis was not attempted and we turned
to a hydroxylation/periodate oxidation procedure. However,
in contrast to the results obtained by Palmer and Jꢃger with
an epimer of 8,[16] bis-hydroxylation with either the OsO4/N-
methylmorpholine-N-oxide (NMO) system or with commer-
cial AD-mix reagent failed. Coinciding with the disappear-
ance of the starting material was the formation of overoxi-
Herein, we describe the crystal structures of some five-
membered iminocyclitols complexed with GH29 a-l-fucosi-
dase from Bacteroides thetaiotaomicron (BtFuc2970) togeth-
er with the synthesis and biological evaluation of ferrocen-
yl–pyrrolidines 5a–c, 14, and their purely organic phenyl an-
alogues 12a,c as both enzyme inhibitors and anticancer
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Iminosugar–Ferrocene Hybrids as Fucosidase Inhibitiors
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Scheme 2. Coupling by reductive amination. a) MgSO4, CH2Cl2;
b) NaBH4, MeOH; c) 1m HCl, MeOH then Amberlyst A-26 (OHꢀ);
d) H2, Pd–C.
Scheme 1. Synthesis of formyl–pyrrolidine: a) vinylMgBr, THF, RT
(83%); b) MsCl, pyr (77%); c) according to ref. [17]; d) Boc2O, NEt3,
RT (97%); e) 60% aq. CH3COOH (69%); f) NaIO4, EtOH/H2O
(100%). Boc=tert-butoxycarbonyl, Ms=methanesulfonyl, pyr=pyridine.
natural a-l-fucosidase substrates and inhibitors 3–5. The
synthesis of 14 used carboxaldehyde 13[12] as the starting ma-
terial and was accomplished following similar reaction se-
quences.
dation and open-chain products, the latter probably resulted
from an intramolecular Cope elimination. Thus, we turned
to an alternative synthesis of the required formyl–pyrroli-
dine, which started with d-mannose diacetonide. A four-step
sequence yielded methylpyrrolidine 9,[17] the stereochemical
configuration of which matched that required for potent in-
hibition of AFU. Using protected 9 as its N-Boc derivative,
selective hydrolysis of the primary acetonide afforded the
expected diol 10 in an acceptable yield (67%). Subsequent
oxidative cleavage with sodium periodate gave the 2-
formyl–pyrrolidine 11 (the enantiomer of which has previ-
ously been prepared using another strategy[18]). Carboxalde-
hyde 11 was used in the coupling procedure without further
purification.
The attachment of a ferrocenylamine moiety to 11 was ac-
complished by using reductive amination (Scheme 2). To
achieve this goal, each amine 6a–c was treated with 11 in
CH2Cl2 in the presence of MgSO4 as a dehydrating agent to
yield the corresponding imine. After filtration and concen-
tration of the reaction mixture, the residue was dissolved in
MeOH and sodium borohydride was added to allow reduc-
tion to the expected amine. Final deprotection of the acid-
labile groups was performed in 1m HCl. Ferrocenyl iminosu-
gars 5a–c were obtained in a pure form (40–64% overall
yield from 11) after neutralization with Amberlyst A-26
(OHꢀ form) and purification by chromatography on silica
gel.
Enzyme inhibition and antiproliferative activity: The fucosi-
dase inhibition, both of the a-l-fucosidase from bovine
kidney and, for selected compounds, the BtFuc2970 enzyme
from Bacteroides thetaiotaomicron as well as the antiproli-
ferative potential (against MDA-MB-231 breast-cancer
cells) of new compounds 5a–c, 12a,c, and 14 were evaluated
and compared to analogues 3a–c and 4a–c (Table 1). Com-
pounds 5a–c displayed Ki values toward bovine kidney AFU
in the sub-micromolar range (Ki =0.19–0.22 mm), thus the
presence of the ferrocene functionality did not impede bind-
ing of the pyrrolidine unit to fucosidase (see below for com-
parison with the non-ferrocenyl analogues).
Ferrocenyliminocyclitols bind to bovine kidney a-l-fucosi-
dase much more tightly than the substrate para-nitrophenyl-
a-l-fucopyranoside (KM =640 mm). Compounds 5a–c were
slightly better inhibitors of AFU than the reported homo-
logues 4a–c (Ki =0.29-0.39 mm). Moreover, in each series, in-
creasing the length of the carbon chain that connects the
pyrrolidine to the ferrocenyl unit (a–c) led to more potent
affinities. This was also observable with the non-ferrocenyl
analogues because 12c (n=3, Ki =0.24 mm) binds more
tightly to AFU than 12a (n=1, Ki =0.57 mm). This result is
in contrast with the data obtained from arylpyrrolidines 3a–
c, in which the presence of the aryl group in proximity to
the fucose surrogate afforded better affinities (Ki =10 nm).
Interestingly, the ferrocenyl-containing derivative 5c (Ki =
0.19 mm) was an even more potent inhibitor than the control
compound 12c (Ki =0.24 mm), an analogue that features a
less sterically demanding phenyl group. Results from Table 1
also show that only the 2S configuration in the pyrrolidine
ring provides an adequate orientation of the C2 “aglycon”
substituent to induce strong binding to AFU, that is, 14
Several analogues were prepared in the same manner
(Scheme 2). Compounds 12a,c, which feature a phenyl
group in place of ferrocene, were isolated after the coupling
of carboxaldehyde 11 with benzylamine or commercial 3-
phenylpropylamine. Furthermore, ferrocenyl iminosugar 14,
the C2 epimer of the known 4c, featured a b-configured
substituent at C2, that is, the opposite stereochemistry to
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J.-B. Behr et al.
Table 1. Fucosidase inhibition and antiproliferative effect (breast-cancer cells)
of aryl– and ferrocenyl–pyrrolidines.[a]
kidney a-l-fucosidase, similar to the analogue 3b (Table 1).
Contrasting results were obtained with both fucosidase sour-
ces; that is, although ferrocenyl iminosugars 4a,c had
weaker affinities than the arylpyrrolidines 3a–c for fucosi-
dase from bovine kidney, they were significantly better li-
gands of BtFuc2970.
Inhibition Inhibition of MDA-
of bovine
kidney
AFU
MB-231
cell growth (% con-
trol)
Inhibitor Structure
Ki [mm]
[I]=50 mm [I]=25 mm
Next, the antiproliferative effect was analyzed by using
the hormone-independent breast-cancer cell-line MDA-MB-
231, which had previously shown to be sensitive to ferrocene
conjugates.[19] Inhibition of cell growth was determined at
two concentrations of the drug (25 and 50 mm) and was eval-
uated by comparison with an untreated control. The antipro-
liferative activity of ferrocenyliminocyclitols 5a–c was simi-
lar to that previously observed for compounds 4a–c depend-
ing on the length (n) of the side chain of the ferrocenyla-
mine moiety (Table 1).[12] No antiproliferative activity was
observed for 5a at the highest concentration, whereas inhib-
ition of 40 and 75% was observed with 5b and 5c, respec-
tively. On the other hand, the number (m) of methylene
groups between the iminosugar and the ferrocenylamine
moieties does not significantly impact the antiproliferative
effect of 5a–c relative to 4a–c. As expected, no antiprolifer-
ative activity was obtained with the phenyl-containing ana-
logues 12a and 12c or with the arylpyrrolidine 3b alone,
thus confirming that only the ferrocenyl moiety is responsi-
ble for the antiproliferative action. Finally, results from
Table 1 also show that the configuration in the pyrrolidine
ring has no effect on the antiproliferative activity of the fer-
rocenyliminocyclitols, with 14 being as active as its 2S coun-
terpart 4c, thus showing there is no direct relationship be-
tween the tightness of fucosidase binding and the antiproli-
ferative activities. Thus, conjugation of the cytotoxic ferroce-
nylamine moiety to the pyrrolidine unit did not decrease its
antiproliferative action. This outcome was also evidenced by
the similarity between the cell-growth curves in presence of
a ferrocenylamine moiety alone and after conjugation to the
pyrrolidine ring (see the Supporting Information).
3a
0.81[c]
nd
nd
nd
ni
3b
0.0095[c]
3c
0.010[c]
0.29
0.39
0.29
0.21
0.22
0.19
0.57
0.24
7.3
nd
nd
4a
4b
4c
ni[b]
ni[b]
45ꢁ19[b]
77ꢁ30[b]
ni
ni[b]
39ꢁ16[b]
5a
5b
5c
ni
40ꢁ17
75ꢁ6
ni
15ꢁ14
42ꢁ19
nd
12a
12c
14
Structural analysis of fucosidase inhibition: A number of 3D
structures for fucosidases from the carbohydrate-active
enzyme (CAZY) family GH29 have previously been deter-
mined including a-l-fucosidases from Thermotoga mariti-
ma,[20] Bacteroides thetaiotaomicron,[11b] and Bifidobacterium
longum.[21] To date, no structural information is available for
mammalian fucosidases and the Bacteroides thetaiotaomi-
cron (BtFuc2970) enzyme can act as a reasonable surrogate
with sequence identity with human GH29 fucosidases
FUCA1 and FUCA2 of 27 and 28%, respectively.
ni
nd
100ꢁ1.5
32ꢁ6
[a] ni=no inhibition, nd=not determined. [b] Taken from ref. [12]. [c] Taken
from ref. [10c].
Crystal structures of BtFuc2970 with 3a–c and 4a,c as li-
gands were obtained (details of the data collection and re-
finement are given in the Supporting Information) to eluci-
date the structural factors that determine the potency of the
inhibition of fucosidase. The presence of inhibitors 3a–c in
their crystal structures was immediately apparent from dif-
ference maps, whereas the presence of 4a and 4c became
apparent after a number of cycles of refinement (Figure 3).
The fuco-configured ring systems of all the inhibitors adopt
(Ki =7.3 mm) was 25-fold less active than its 2S counterpart
4c toward fucosidase.
Prior to structural analysis, tight binding to the bacterial
enzyme was confirmed. The inhibition of BtFuc2970 by 3a,b
and 4a,c (Ki =5.4, 3.5, 0.52, and 0.46 mm, respectively) was
studied (see the Supporting Information). Unfortunately, in-
sufficient compound was available to determine a Ki value
for 3c, which displayed potent inhibition toward bovine-
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Iminosugar–Ferrocene Hybrids as Fucosidase Inhibitiors
FULL PAPER
fraction data for BtFuc2970
with 4a as a ligand at l=1.2 ꢄ
and a significant “anomalous”
signal was observed (Figure 4).
The ferrocenyl “aglycons” inter-
act through Van der Waalsꢁ
forces, which has been observed
previously
in
ferrocenyl
enzyme/inhibitor complexes.[22]
The fact that the ferrocenyl
moieties of 4a,c point toward
the solvent molecules rather
than being buried in a hydro-
phobic pocket (as do the ferro-
cene complexes in ref. [22]) ex-
plains the high temperature-fac-
tors observed. The exocyclic
amine that tethers the ferro-
cene and pyrrolidine groups is
invariantly coordinated to a sul-
fate moiety likely abstracted
from the crystallization liquor
(Figure 4).
Figure 3. Inhibitors a) 3a, b) 3b, c) 3c, d) 4a, and e) 4c that lie in the BtFuc2970 active site. Maps shown are
Fo–Fc maps contoured at 5 s (3a–c) or 3 s (4a,c), in each case calculated by using phases prior to the incorpo-
ration of the ligand in refinement. The enzymatic acid/base and nucleophile are shown and labeled in (a).
The small-molecule crystal
3E envelope conformations. The “aglycon” moieties of these
inhibitors lay atop a hydrophobic ridge formed by residues
tryptophan 88 (Trp88) and Trp232 and point toward the sol-
vent molecules in contrast to the “aglycons” for six-mem-
bered inhibitors.[11b]
Compound 3a has an N-methylated endocylic amine and
displays only a slight difference in binding affinity relative
to its parent compound 3b, which lacks N-methylation (Ki =
5.4 vs. 3.5 mm), thus indicating that BtFuc2970 can easily ac-
commodate the extra steric bulk. This finding can be ob-
served in the crystal structures, with only minimal differen-
ces observed between the structures of 3a and 3b. The N-
methyl group of 3a disrupts a hydrogen-bonding interaction
that exists between a water molecule and the catalytic nucle-
ophile aspartic acid 229 (Asp229) in the complex with 3b
(see the Protein Data Bank (PDB), entry 4JFT). Additional-
ly, arginine 262 (Arg262) is slightly displaced toward the cat-
alytic acid/base in the complex
structure of 3a has already been determined.[10c] Interesting-
ly, the conformations observed for 3a in both the free-state
and enzyme-bound crystal structures appear to be almost
identical (0.18 ꢄ root-mean square deviation; see the Sup-
porting Information). Hence, binding of pyrrolidines, such
as 3, occurs without energy-demanding conformational dis-
tortion of the ligand to match the shape of the enzyme cata-
lytic site, which could explain, at least in part, the strong af-
finities of our five-membered fucose mimics for fucosidases.
Conclusion
Five-membered iminosugars display potent affinities for fu-
cosidases. This particular property could be exploited for
the selective delivery of antitumor drugs toward fucosidase-
rich malignant tissues. We prepared a series of iminosugar–
with 3a (see the PDB, en-
try 4JFS) and forms hydrogen
bonds with this residue. N-
Methylation seems much more
detrimental for the binding
with bovine-kidney protein be-
cause the Ki value increases
from 10 nm to 3.5 mm for 3b and
alkylated 3a, respectively.
The ferrocenyl “aglycon”
moieties of 4a and 4c are
highly disordered. The presence
of the ferrocene group in the
crystal structure was confirmed
Figure 4. Surface representation, in divergent (“wall-eyed”) stereo of the active site of BtFuc2970 with 4c. The
inhibitor atoms are displayed as cylinders (carbon atoms in gray, others colored by atom type). The map
shown is an anomalous difference map (averaged over the unit cell) contoured at 5 s. The protein atoms of
through the collection of dif- BtFuc2970 from 4JFW are displayed as a white surface.
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J.-B. Behr et al.
Compound 8 (77%, yellow oil): Rf =0.87 (Et2O/petroleum ether 3:7);
[a]D20 = +7.1 (c=0.54 in CHCl3); IR (film): n˜max =878, 1000, 1063, 1123,
ferrocene hybrids, which retained both the fucosidase inhibi-
tion capability of the iminocyclitol moiety and the cytotoxic-
ity of the ferrocenylamine species. Comparison with control
analogues, which either lack the ferrocenyl functionality or
bind much less tightly to the AFU, clearly showed the pyrro-
lidine unit to have no cytotoxic effect on its own, with all
the antiproliferative activity being potentiated by the ferro-
cenyl moiety. Little is known about the structural basis for
AFU inhibition by polyhydroxypyrrolidines. Crystal struc-
tures of a number of five-membered iminocyclitols bound to
a-l-fucosidase BtFuc2970 were obtained to identify the fac-
tors that determine the potency of binding. These structures
1160, 1209, 1378, 1454, 2806, 2933, 2984 cmꢀ1 1H NMR (250 MHz,
;
CDCl3): d=1.13 (d, 3JHH =6.3 Hz, 3H; 6-H), 1.34 (s, 3H; iPr), 1.57 (s,
3H; iPr), 2.90–3.08 (m, 1H; 5-H), 3.38–3.57 (m, 2H; 2-H and CH2-Ph),
3.82 (d, 2JHH =14.1 Hz, 1H; CH2-Ph), 4.40–4.43 (m, 1H; 3-H), 4.61 (t,
3JHH =5.7 Hz, 1H; 4-H), 5.12 (dd, JHH =50.0, 13.7 Hz, 2H; 2’-H), 5.62–
5.82 (m, 1H; 1’-H), 7.14–7.40 ppm (m, 5H; Ar); 13C NMR (62.5 MHz,
CDCl3): d=12.45 (6-C), 25.59 and 26.43 (iPr), 50.45 (CH2-Ph), 58.59 (5-
C), 68.85 (2-C), 81.74 (4-C), 83.79 (3-C), 119.39 (2’-C), 126.48, 128.04 and
128.24 (Ar), 133.60 ppm (1’-C); HRMS (ESI): m/z calcd for C17H23NO2:
274.1807 [M+H+]; found: 274.1810.
Synthesis of aldehyde 11: Boc2O (459 mg, 2 mmol, 1.1 equiv) was added
in small portions to methylpyrrolidine 9[17] (344 mg, 1.338 mmol) in
CH2Cl2 (5 mL) and Et3N (470 mL, 4.014 mmol, 3 equiv) at 08C. The reac-
tion mixture was allowed to warm slowly to room temperature and was
stirred for additional 24 h. Distilled water (20 mL) was added to the mix-
ture, and the separated aqueous layer was extracted with dichlorome-
thane (3ꢅ20 mL). The combined organic layers were dried (MgSO4), fil-
tered, and evaporated under reduced pressure. The crude residue was pu-
rified by column chromatography on silica gel (EtOAc/petroleum ether
1:9) to afford the N-Boc derivative (462 mg, 97%,) as a white solid.
3
revealed that pyrrolidines adopt a E envelope conformation
in the active site, thus mimicking the proposed
3H4 conformation for the catalytic transition state. It is this
conformation that the inhibitor also adopts in its small-mol-
ecule crystal structure. Interestingly, the ferrocenyl moiety
of the ferrocene-containing inhibitors points toward the sol-
vent molecules, being available for the generation of ROS
by being in contact with the biological environment in vivo.
In the near future, we intend to evaluate the ability of our
compounds to cross cancer tissues selectively in vivo to lay
the foundations of such a therapeutic approach.
This protected pyrrolidine (153 mg, 0.428 mmol) in 60% aq. CH3COOH
(5 mL) was stirred 15 h at room temperature. The volatiles were evapo-
rated under reduced pressure and after purification by column chroma-
tography on silica gel (petroleum ether/EtOAc 4:6) the expected diol 10
(94 mg, 69%) was obtained as a yellow oil.
Diol 10 (69%, yellow oil): [a]D20 = +44.9 (c 0.79, CDCl3); 1H NMR
(250 MHz, CHCl3): d=1.32 (s, 6H; iPr, CH3), 1.45 (s, 12H; iPr, Boc),
3.35–3.59 (m, 2H; 2’-H), 3.81–3.98 (m, 2H; 5-H and 1’-H), 4.15–4.30 (m,
1H; 2-H), 4.51–4.67 ppm (m, 2H; 3-H and 4-H); 13C NMR (63 MHz
CDCl3): d=16.11 (CH3), 25.18, 26.24, 28.46 (2ꢅiPr and Boc), 58.27 (5-
C), 63.46 (2’-C), 63.82 (2-C), 73.60 (1’-C), 81.14, 81.55 ppm (3-C and 4-
C); HRMS (ESI): m/z calcd for C15H27NO6: 340.1736 [M+Na]+; found:
340.1744.
Experimental Section
General: All the reactions were performed under argon. The reagents
and solvents were commercially available in high purity and used as re-
ceived. Silica gel F254 (0.2 mm) was used for the TLC plates and detec-
tion was carried out by spraying with an alcoholic solution of phospho-
molybdic acid, para-anisaldehyde, or an aqueous solution of KMnO4
(2%)/Na2CO3 (4%), followed by heating. Flash column chromatography
was performed over silica gel M 9385 (40–63 mm) Kieselgel 60. NMR
spectra were recorded on Bruker AC250 (250 and 62.5 MHz for 1H and
13C nuclei, respectively) or 600 (600 and 150 MHz for 1H and 13C nuclei,
respectively) spectrometers. Chemical shifts are expressed in parts per
million (ppm) and were calibrated to the residual solvent peak. Coupling
constants are in Hz and the splitting pattern abbreviations are br=broad,
s=singlet, d=doublet, t=triplet, q=quartet, qt=quintuplet, and m=
multiplet. IR spectra were recorded with an IR plus MIDAC spectropho-
tometer and are expressed in cmꢀ1. Optical rotations were determined at
208C with a Perkin–Elmer Model 241 polarimeter in the specified sol-
vents. High-resolution mass spectrometry (HRMS) was performed on Q-
TOF Micro micromass positive ESI (CV=30 V).
NaIO4 (170 mg, 0.797 mmol, 2.7 equiv) was added to diol 10 (94 mg,
0.305 mmol) in EtOH/water (6 mL, 2:1). The solution was stirred at
room temperature for 1 h, then water (10 mL) and dichloromethane were
added. After separation of the layers, the aqueous phase was washed
with dichloromethane (3ꢅ10 mL). The combined organic layers were
dried (MgSO4), filtered, and evaporated to afford the crude aldehyde 11
(85 mg, 100%, yellow oil), which was used in the next step without fur-
ther purification.
General procedure for the synthesis of hybrids 5, 12, and 14: MgSO4
(119 mg, 1.00 mmol, 10 equiv) and ferrocenylamine 5 (1.2 equiv) were
successively added to aldehyde 11 (35 mg, 0.100 mmol) in dichlorome-
thane (1.5 mL). The solution was stirred at room temperature for 5 h.
The solution was filtered and concentrated. NaBH4 (5 mg, 0.130 mmol,
1.3 equiv) was added to the resulting material dissolved in MeOH (2 mL)
at 08C. This solution was stirred and left to warm to room temperature
overnight. A saturated solution of NH4Cl and EtOAc were successively
added at 08C, and the resulting organic layer was separated. The aqueous
layer was extracted with EtOAc, and the combined organic layers were
dried over MgSO4 and concentrated. The residue was purified by chro-
matography on silica gel (EtOAc) to yield pure the ferrocenyl iminosu-
gar.
Synthesis of vinylpyrrolidine 8: Vinylmagnesium bromide (30.6 mL of a
commercial 1m solution, 30.6 mmol, 4 equiv) was added dropwise to a
stirred solution of glycosylamine 7 (1.990 g, 7.66 mmol) in THF (20 mL)
at 08C. The resulting mixture was left at room temperature for 7 h. Satu-
rated NH4Cl was added to the reaction mixture, and the solution was ex-
tracted with Et2O (3ꢅ20 mL). The combined organic layers were dried
(MgSO4) and evaporated. Purification by flash column chromatography
(EtOAc/petroleum ether 4:6) yielded the intermediate aminoalcohol
(Rf =0.43, 1.162 g, 52%) as a yellow oil.
The solution of ferrocenyl iminosugar (0.042 mmol) in MeOH (1 mL)
was treated with 3m HCl (1 mL). The mixture was stirred at room tem-
perature overnight. After completion of the reaction the solution was
neutralized with Amberlyst A-26 (OHꢀ) and evaporated. Purification by
column chromatography on silica gel (dichloromethane/MeOH 8:2!
CHCl3/MeOH/1m NH4OH 6:4:1) yielded ferrocenyl iminosugar 5 as a
yellow film.
MsCl (640 mL, 8.175 mmol, 2.45 equiv) was added dropwise to a solution
of pure aminoalcohol (1.082 g, 3.715 mmol) in pyridine (4.7 mL) and
THF (4.7 mL) at 08C. The mixture was stirred for 2 h at 08C and for an
additional 2 h at room temperature. Saturated solutions of NH4Cl and
Et2O were successively added at 08C, and the resulting organic phase
was separated. The aqueous layer was extracted with Et2O and the com-
bined organic layers were dried over MgSO4 and concentrated. The resi-
due was purified by chromatography on silica gel (Et2O/petroleum ether
1:9) to yield pure allylpyrrolidine 8 (783 mg, 77%) as a yellow oil.
Compound 5a (64% from 10): [a]D20 =ꢀ25.0 (c=0.76 in MeOH);
1H NMR (600 MHz, MeOD): d=1.21 (d, 3JHH =6.7 Hz, 3H; CH3), 2.71
(dd, 3JHH =12.3, 8.6 Hz, 1H; 1’-H), 2.86 (dd, 3JHH =12.3, 4.7 Hz, 1H; 1’-
3
3
H), 3.22 (qd, JHH =6.7, 2.9 Hz, 1H; 2-H), 3.27 (td, JHH =8.2, 4.7 Hz, 1H;
3
5-H), 3.64 (s, 2H; 2’-H), 3.81–3.84 (m, 1H; 4-H), 3.86 ppm (dd, JHH =7.7,
&
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www.chemeurj.org
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

Iminosugar–Ferrocene Hybrids as Fucosidase Inhibitiors
FULL PAPER
4.1 Hz, 1H; 3-H); 13C NMR (63 MHz, MeOD): d=12.82 (CH3), 49.24,
49.62 (1’,2’-C), 57.82, 60.93 (2,5-C), 69.73, 70.57 (Fc), 74.00 (4-C), 76.7 (3-
C), 81.63 ppm (Cq-Fc) HRMS (ESI): m/z calcd for C17H24FeN2O2:
345.1265 [M+H]+; found: 345.1263.
off equation. All the assays were done in duplicate (less than 10% varia-
bility in each case).
BtFuc2970: Substrate 2-chloro-4-nitrophenyl-a-l-fucopyranoside (CNP-
fucoside) was purchased from Carbosynth Ltd. The experiments were
run over a time course of 5 min during which absorbance at l=405 nm
was detected. For each data point, a solution of 50 mm HEPES buffer
(pH 700 mm NaCl 250 nm BtFuc2970 was equilibrated thermally (378C)
in the presence of a varying concentration (inhibitor concentrations
straddling the Ki value) of the inhibitor. To each of these solutions 50 mm
CNP-fucoside was added to initiate hydrolysis. The Ki values were deter-
mined by analyzing the initial enzyme rates in the absence of inhibitor
and comparing with the rate in the presence of increasing concentrations
of inhibitor. The Ki value were calculated as the reciprocal of the gradi-
ent of each plot.
Compound 5b (40% from 10): [a]D20 =ꢀ11.5 (c=0.4 in MeOH);
1H NMR (600 MHz, D2O): d=1.14 (d, 3JHH =6.7 Hz, 3H; CH3), 2.52 (t,
3JHH =7.6 Hz, 2H; NCH2CH2Fc), 2.68 (dd, 3JHH =12.3, 8.6 Hz, 1H; 1’-H),
3
2.73–2.79 (m, 2H; NCH2 CH2Fc), 2.81 (dd, JHH =12.3, 4.4 Hz, 1H; 1’-H),
3.17 (qd, 3JHH =6.4, 2.7 Hz, 1H; 5-H), 3.20–3.22 (m, 1H; 2-H), 3.72–3.78
(m, 1H; 4-H), 3.81 ppm (dd, 3JHH =7.8, 4.0 Hz, 1H; 3-H); 13C NMR
(63 MHz, D2O): d=13.88 (CH3), 30.21 (NCH2CH2Fc), 51.58
(NCH2CH2Fc), 52.80 (1’-C), 56.91 (5-C), 61.69 (2-C), 68.50, 69.26, 69.56
(3ꢅFc), 74.91 (4-C), 77.91 (3-C), 86.89 ppm (Cq-Fc); HRMS (ESI): m/z
calcd for C18H26FeN2O2: 359.1422 [M+H]+; found: 359.1432.
Compound 5c (59% from 10): [a]D20 =ꢀ20.9 (c=0.38 in MeOH);
1H NMR (600 MHz, MeOD): d=1.29 (d, 3JHH =6.7 Hz, 3H; CH3), 1.77–
1.88 (m, 2H; CH2CH2CH2Fc), 2.39–2.49 (m, 2H; CH2CH2CH2Fc), 2.86
(qt, JHH =11.9, 7.4 Hz, 2H; CH2CH2CH2Fc), 2.95 (dd, JHH =12.9, 9.1 Hz,
1H; 1’-H), 3.09 (dd, 3JHH =12.9, 4.2 Hz, 1H; 1’-H), 3.43 (m, 2H; 2,5-H),
3.89–3.93 (m, 1H; 3-H), 3.99 (dd, 3JHH =8.0, 3.8 Hz, 1H; 4-H), 4.05–
4.15 ppm (m, 9H; Fc); 13C NMR (151 MHz, MeOD): d=13.04 (CH3),
Antiproliferative assays
Cell culture: The breast-cancer cell line MDA-MB-231 was obtained
from the American Type Cell Culture Collection (Rockville, MD, USA).
Cell culture reagents were purchased from Lonza (Levallois-Perret,
France). Cells were routinely grown in monolayers and maintained at
378C in an atmosphere of 5% CO2, in the Dulbecco modified eagle
medium (DMEM) supplemented with 10% fetal bovine serum (FBS),
2 mm l-glutamine, 100 unitsmLꢀ1 penicillin–streptomycin.
3
3
27.96
(NCH2CH2CH2Fc),
30.55
(NCH2CH2CH2Fc),
49.79
(NCH2CH2CH2Fc), 50.84 (1’-C), 57.68 (5-C), 60.99 (2-C), 68.34, 69.06,
69.52 (3ꢅFc), 74.25 (3-C), 76.93 (4-C), 89.10 ppm (Cq-Fc); HRMS (ESI):
m/z calcd for C19H28FeN2O2: 373.1578 [M+H]+; found: 373.1571.
Proliferation assays: An aliquot of 2ꢅ103 cells were seeded in 96-well
plates in DMEM, 10% FBS. The medium was replaced after 24 h, and
the cells were treated for 48 h with 0, 25, and 50 mm of iminosugars. Cell
numbers were determined by adding 3-(4,5-dimethylthiazol-2-yl)-5-(3-car-
boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) to the
wells 2 h before spectrophotometric reading (absorbance at l=490 nm).
The results are expressed for each concentration of inhibitor for the con-
trol as mean value of 8 wellsꢁSD.
Compound 12a (57% from 10): [a]D20 =ꢀ0.05 (c=0.2 in MeOH);
3
1H NMR (600 MHz, D2O): d=1.12 (d, JHH =6.7 Hz, 3H; CH3), 2.67 (dd,
3JHH =12.3, 8.9 Hz, 1H; 1’-H), 2.81 (dd, 3JHH =12.3, 4.4 Hz, 1H; 1’-H),
3.18 (qd, 3JHH =6.6, 2.8 Hz, 1H; 5-H), 3.24 (td, 3JHH =8.3, 4.4 Hz, 1H; 2-
H), 3.81 (s, 2H; NCH2Ph), 3.89–3.94 (m, 2H; 3 and 4-H), 7.30–7.50 ppm
(m, 5H; Ar); 13C NMR (63 MHz, D2O): d=13.49 (CH3), 51.83 (1’-C),
52.89 (NCH2Ph), 55.04 (5-C), 60.01 (2-C), 74.26, 76.93 (3 and 4-C),
128.01, 129.15 ppm (3ꢅAr); HRMS (ESI): m/z calcd for C13H20N2O2:
237.1603 [M+H]+; found: 237.1602.
Compound 12c (44% from 10): [a]D20 =ꢀ28.0 (c=0.5 in MeOH);
Acknowledgements
3
1H NMR (500 MHz, D2O): d=1.14 (d, JHH =6.7 Hz, 3H; CH3), 1.88–1.97
3
(m, 2H; NCH2CH2CH2Fc), 2.71 (t, JHH =7.6 Hz, 2H; NCH2CH2 CH2Fc),
This work was supported by the University of Sciences and Technologies
of Lille, the Association pour la Recherche sur le Cancer (Grant no. 7936
and 5023), the comitꢀ de lꢁAisne de La Ligue contre le Cancer, and the
Ministꢆre de lꢁEnseignement Supꢀrieur et de la Recherche (PhD student-
ship to A.H.). This work was also supported by the Biotechnology and
Biological Sciences Research Council (BBSRC) through a PhD student-
ship to D.W.W. This work was carried out with the support of the Dia-
mond Light Source. We thank Garib Murshudov for the generation of
stereochemical target values and coordinate sets for ferrocenyl inhibitors
4a,c.
2.78–2.87 (m, 3H; N CH2CH2CH2Fc and 1’-H), 2.97 (dd, 3JHH =12.6,
3
4.3 Hz, 1H; 1’-H), 3.20 (qd, 3JHH =6.7, 2.9 Hz, 1H; 5-H), 3.28 (td, JHH
=
8.6, 4.3 Hz, 1H; 2-H), 3.90–3.94 (m, 1H; 4-H), 3.97 (dd, 3JHH =8.1,
4.1 Hz, 1H; 3-H), 7.25–7.34 (m, 3H; Ar), 7.37–7.43 ppm (m, 2H; Ar);
13C NMR (62 MHz, D2O): d=13.27 (CH3), 28.65 (NCH2CH2CH2Fc),
32.58 (NCH2CH2CH2Fc), 48.25 (NCH2CH2CH2Fc), 51.51 (1’-C), 55.38 (5-
C), 58.70 (2-C), 74.05 (4-C), 76.69 (3-C), 126.73, 128.91, 129.15ppm (3ꢅ
Ar); HRMS (ESI): m/z calcd for C15H25N2O2: 265.1916 ([M+H]+; found:
265.1911.
Compound 14 (69% from 13): [a]D20 = +0.4 (c=1 in MeOH); 1H NMR
(600 MHz, MeOH): d=1.38 (d, 3JHH =6.8 Hz, 3H; CH3), 1.86–1.99 (m,
3H; NCH2CH2CH2Fc), 2.10 (dq, 3JHH =8.6, 6.7 Hz, 1H; 1’a-H), 2.28
(ddd, 3JHH =15.2, 11.3, 6.7 Hz, 1H; 1’b-H), 2.43–2.51 (m, 2H;
NCH2CH2CH2Fc), 2.98–3.06 (m, 2H; NCH2CH2CH2Fc), 3.10–3.23 (m,
2H; 2’-H), 3.54–3.61 (m, 1H; 5-H), 3.62–3.68 (m, 1H; 2-H), 4.20 (t,
3JHH =4.8 Hz, 41H; -H), 4.36 ppm (dd, 3JHH =6.2, 4.8 Hz, 1H; 3-H);
13C NMR (151 MHz, MeOD): d=12.78 (CH3), 25.54 (1’-C), 27.59
(NCH2CH2CH2Fc), 28.92 (NCH2CH2CH2Fc), 45.93 (2’-C), 57.85 (5-C),
59.05 (2-C), 68.46, 69.05, 69.57 (3xFc), 71.93 (3-C), 72.57 (4-C),
88.32 ppm (Cq-Fc); HRMS (ESI): calcd for C20H30FeN2O2: 387.1735
[M+H]+; found: 387.1722.
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Received: March 15, 2013
Published online: && &&, 0000
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www.chemeurj.org
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
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These are not the final page numbers!

Iminosugar–Ferrocene Hybrids as Fucosidase Inhibitiors
FULL PAPER
Glycosidase Inhibitors
A. Hottin, D. W. Wright,
A. Steenackers, P. Delannoy, F. Dubar,
C. Biot, G. J. Davies,
J.-B. Behr* .................... &&&& — &&&&
a-l-Fucosidase Inhibition by Pyrroli-
dine–Ferrocene Hybrids: Rationaliza-
tion of Ligand-Binding Properties by
Structural Studies
Transition-state-mimicking 3E confor-
mations (see picture) are evident from
the three-dimensional structures of fer-
rocenyl iminosugar/fucosidase com-
plexes. Novel pyrrolidine–ferrocene
conjugates show strong anti-fucosidase
and antiproliferative action, with up to
100% inhibition of proliferation of an
MDA-MB-231 cancer-cell line at
50 mm.
Chem. Eur. J. 2013, 00, 0 – 0
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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These are not the final page numbers!