Galectin-inhibitory thiodigalactoside ester derivatives have antimigratory effects in cultured lung and prostate cancer cells

Article abstract of DOI:10.1021/jm801077j

Aromatic 3,3?-diesters of thiodigalactoside were synthesized in a rapid three-step sequence from commercially available thiodigalactoside and evaluated as inhibitors of cancer- and immunity-related galectins. For each of galectins-1, -3, -7, and -9N-terminal domain, aromatic 3,3′-diesters of thiodigalactoside were found to have affinities in the low micromolar range, which represents a 7-70 fold enhancement over thiodigalactoside itself. No significant improvement was found for galectin-8 N-terminal domain. Two of the compounds were selected for testing in cell culture and were shown to have potent antimigratory effects on human PC-3 prostate and human A549 nonsmall-cell lung cancer cells.

Full text of DOI:10.1021/jm801077j

J. Med. Chem. 2008, 51, 8109–8114
8109
Galectin-Inhibitory Thiodigalactoside Ester Derivatives Have Antimigratory Effects in Cultured
Lung and Prostate Cancer Cells
Tamara Delaine,† Ian Cumpstey,† Laurent Ingrassia,‡ Marie Le Mercier,| Paul Okechukwu,† Hakon Leffler,*,§ Robert Kiss,*,|
and Ulf J. Nilsson*,†
Organic Chemistry, Lund UniVersity, POB 124, SE-221 00 Lund, Sweden, Unibioscreen SA, 40 AVenue Joseph Wybran 1070, Brussels,
Belgium, Section MIG, Department of Laboratory Medicine, Lund UniVersity, So¨lVegatan 23, SE-223 62 Lund, Sweden, Laboratoire de
Toxicologie, Institut de Pharmacie, UniVersite´ Libre de Bruxelles, Brussels, Belgium
ReceiVed August 29, 2008
Aromatic 3,3′-diesters of thiodigalactoside were synthesized in a rapid three-step sequence from commercially
available thiodigalactoside and evaluated as inhibitors of cancer- and immunity-related galectins. For each
of galectins-1, -3, -7, and -9N-terminal domain, aromatic 3,3′-diesters of thiodigalactoside were found to
have affinities in the low micromolar range, which represents a 7-70 fold enhancement over thiodigalactoside
itself. No significant improvement was found for galectin-8 N-terminal domain. Two of the compounds
were selected for testing in cell culture and were shown to have potent antimigratory effects on human
PC-3 prostate and human A549 nonsmall-cell lung cancer cells.
Chart 1. Structure of Thiodigalactoside 1, C3′-Modified
LacNAc Derivatives 2, C2-Esters of Lactose 3, and
Di-amido-thiodigalactosides 4
Introduction
The galectins, a family of ꢀ-galactoside-binding proteins, have
been implicated in numerous biological activities1-11 including
regulation of apoptosis,12,13 intracellular trafficking,14 cell
signaling,15 and cell adhesion.11,16 These activities in turn play
important roles in inflammation,3,17-20 immunity,21 cancer
progression,4,5,8,16,22,23 and, in direct relation to the aim of the
present work, cancer cell migration.24-26
Recently, insight into their molecular mechanisms of actions
has deepened, as exemplified by the T-cell cell surface protein
glycosylation patterns controlling sensitivity to galectin-1
induced apoptosis,27 galectin-8 ligand specificities regulating
its cell sorting,28,29 and by the discovery that galectin-3 induced
lattice formation with branched N-glycans regulates cell surface
receptor trafficking.30-32 Several of these discoveries point to
galectins being attractive therapeutic targets in certain clinical
situations, including the fact that galectin-133,34 and galectin-
3,35 for example, are implicated in cancer cell resistance to
chemotherapy. Hence, the development of high-affinity inhibi-
tors showing selectivity for individual members of the galectin
family of proteins has emerged as an important task.36,37
Moreover, it is highly desirable to have convenient access to
high-potency galectin inhibitors.
The unnatural disaccharide thiodigalactoside 1 (Chart 1) binds
to the galectins and has been shown to interact with galectin-1
with a binding mode and conformation similar to that of
LacNAc.38 These observations, combined with our discoveries
that modification of the 3′-position of LacNAc 239,40 or
2-position of lactose 341 leads to high potency inhibitors of
galectins, led us to synthesize the C2-symmetrical diamido-
thiodigalactoside derivatives 4 (Chart 1).42,43 However, while
the synthesis of the precursor compounds may be achieved on
a multigram scale in good overall yield, the synthesis of the
inhibitors 4 involves a large number of steps.
Thiodigalactoside 1 is commercially available or, alterna-
tively, can easily be synthesized from galactose.44 Hence,
exploiting this readily available starting material for a potentially
simpler three-step synthesis of ester analogues of the diamido-
thiodigalactosides 4 emerged as an attractive alternative. Herein,
we detail the preparation of thiodigalactoside esters and their
binding properties to galectin-1, -3, -7, -8N (N-terminal domain),
and -9N (N-terminal domain). Furthermore, two thiodigalacto-
* Corresponding authors: Phone: +4646173270 (H.L.); +32477622083
(R.K.); +46462228218 (U.J.N). Fax: +4646137468 (H.L.); +3223325335
(R.K.); +46462228209 (U.J.N.). E-mail: hakon.leffler@med.lu.se (H.L.);
rkiss@ulb.ac.be (R.K.); ulf.nilsson@organic.lu.se (U.J.N.).
†
Organic Chemistry, Lund University.
Unibioscreen SA.
Laboratoire de Toxicologie, Institut de Pharmacie, Universite´ Libre de
‡
|
Bruxelles.
§
Section MIG, Department of Laboratory Medicine, Lund University.
Abbreviations: NSCLC, nonsmall-cell-lung cancer; HRPC, hormone-
a
refractory prostate cancer; BCNU, carmustine.
10.1021/jm801077j CCC: $40.75
2008 American Chemical Society
Published on Web 11/21/2008

8110 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Delaine et al.
Scheme 1a
Scheme 2a
a Reagents and conditions: (a) HOBt ester, CH2Cl2, Et3N. (b) Dowex
50XB, MeOH.
a Reagents and conditions: (a) PhCH(OMe)2, CSA, DMSO, 60 °C. (b)
BzCl (4.0 equiv), pyridine, 0 °C; 7, 25%; 9, 40%; 10, 16%; OR BzCl (3.0
equiv), pyridine, 0 °C; 6, 18%; 7, 22%; 8, 17%; (c) Dowex 50XB, MeOH.
amide functionalities, as galectin-3 and 7 preferred amides
4a-c43 over the corresponding esters 11, 16, and 19 with more
than 1-2 orders of magnitude and galectin-8N showed a
significant preference for the esters 11, 16, and 19 over the
amides 4a-c.
Structural preferences for the aromatic groups of the esters
11, 16, and 19 paralleled those observed for the amides 4a-c
(Chart 2), suggesting that the aromatic groups of the esters
interact with galectin-1, -3, -7, and -9N in a manner similar to
the aromatic groups of the amides. Hence, the differences
between esters and amides observed for galectin-3, -7, and -9N
may be attributed to the ester and amide functionalities.
However, esters 11 and 16-19 all displayed affinity for galectin-
8N similar to that of thiodigalactoside 1 itself, suggesting that
the aromatic groups do not interact favorably with this galectin
but rather do not interfere with binding as the corresponding
amides 4a-c do.
Compounds 16 and 18 were selected for further evaluation
in a cell-based tumor motility assay49 because they exhibit the
best inhibitory effects for all the galectins tested with affinities
in the range of Kd ∼ 0.7-42 µM (Table 1). The manner in
which we quantitatively determined the levels of cell motility
by means of computer-assisted phase-contrast microscopy
(quantitative video microscopy) has been fully described.50,51
We made use of the human A549 nonsmall-cell-lung cancer
(NSCLCa) and the human PC-3 hormone-refractory prostate
cancer (HRPC) models because these two models express
galectins and display high levels of cell motility in vitro.51
Human A549 expresses at least galectin-152 and human PC-3
HRPC expresses galectins-1, -3, and -8 (R. Kiss; unpublished
data).
Measurements were made at three different inhibitor con-
centrations: 1, 10, and 100 nM and the effect on migration
measured after 12 and 24 h for each inhibitor-cell-line
combination (Figure 1). The data show that compound 16
displayed significant inhibitory effects on A549 NSCLC and
PC-3 HRPC cells, with the highest observed effect at 10 nM
for 24 h in A549 NSCLC cells and at 100 nM for 24 h in PC-3
HRPC cells. The highest observed inhibitory effect of compound
18 on A549 NSCLC cell motility was seen at 100 nM for 24 h,
while on PC-3 HRPC cells, it was at 1 nM for 24 h.
side esters were identified as low micromolar inhibitors of the
galectins studied. These thiodigalactoside esters were also shown
to possess efficient antimigratory effects on experimental lung
and prostate tumor cells.
Chemistry. The 4- and 6-positions of thiodigalactoside 1 were
protected as benzylidene acetals using dimethoxytoluene and
catalytic acid. Starting material 1 and product 5 were insoluble
in DMF or acetonitrile resulting in moderate yields of the
benzylidene-protected 5. Fortunately, high temperatures in
DMSO dissolved both 1 and 5, which resulted in good yield of
5 (Scheme 1). With the 4,6-protected compound 5 in hand,
attention turned to a possible selective protection of the
3-position using acylating reagents.
Treatment of 5 with benzoyl chloride (4.0 equiv) in pyridine
gave a mixture of 3,3′-dibenzoate 7, 2,3,3′-tribenzoate 9, and
2,2′,3,3′-tetrabenzoate 10. With a smaller excess of benzoyl
chloride (3.0 equiv), once again a mixture was obtained, this
time of the 3-monobenzoate 6, 3,3′-dibenzoate 7, and 2′,3-
dibenzoate 8. Apparently, the 3-positions are somewhat more
reactive than the 2-positions toward benzoyl chloride but not
enough to ensure high regioselectivity in the benzoylations.
Hence, an investigation of methods for attenuating the acylating
reagent reactivity was deemed necessary to improve the regi-
oselectivity. Benzoylation in the presence of pyridine and
45
NiCl2 significantly improved the regioselectivity, as did
moderating the reactivity of the benzoylating reagent by
converting the benzoyl chloride to the corresponding N-
hydroxybenzotriazole ester.46 Benzoylation with N-hydroxy-
benzotriazole esters was established as the method of choice
because it proved to have the best reproducibility. Hence,
acylations with N-hydroxybenzotriazole esters, followed by
benzylidene acetal hydrolysis, a series of substituted 3,3′-
diesters, 11 and 16-19, was obtained (Scheme 2).
Biology. In vitro evaluation of dissociation constants in a
fluorescence polarization assay47,48 revealed that galectin-1, -3,
-7, and -9N all displayed improved binding for 11 and 16-19
over the parent thiodigalactoside 1 (Table 1 and Chart 2), clearly
evidencing the involvement of the aromatic ester moieties in
favorable interactions with these galectins. Only one ester, 18,
inhibited galectin-8N significantly better than the parent thio-
digalactoside 1. In general, galectin-1 and -9N did not display
strong discrimination between esters or amides (compare 11 and
4a, 16 and 4b, and 19 and 4c). In contrast, binding to the
remaining galectins was clearly influenced by the ester and
The effects on cell migration of 16 and 18 are observed at
concentrations well below those required to inhibit any galectin
in the binding assay. One possible explanation is that as the
inhibitors accumulate in the cells and achieve high enough local
concentrations, they are able to inhibit galectin activity. Another

Antimigratory Thiodigalactoside Esters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8111
Table 1. Kd Values (µM) against Galectin-1, -3, -7, -8N, and -9N as
compounds 16 and 18 display different binding affinities for
different galectins and (ii) A549 NSCLC and PC-3 HRPC cells
display different patterns of galectin expression. The fact that
these two cancer cell lines display different patterns of galectin
expression implies that the binding of antigalectin compounds
(as for example compounds 16 and 18) will activate different
signaling pathways in these cancer cells, which in turn will
impact cancer cell motility differently.11,16,24,25 Hence, one
hypothesis may be that at lower inhibitor concentrations
inhibition of a tighter-binding galectin dominates, which via
effects on certain signaling pathways confers antimigratory
effects. At higher inhibitor concentrations, less strongly inhibited
galectin(s) are affected, which may influence other signaling
pathways resulting in increased motility and hence a bell-shaped
curve. Antimigratory effects of a given compound often occur
as a bell-shaped curve with an optimum at a specific concentra-
tion.53 This feature has been well documented for neuropeptides
as potential antimigratory nontoxic anticancer agents.54,55 With
respect to the aim of the current study, i.e., developing inhibitors
against galectins that are implicated in cancer cell migration, it
has already been demonstrated for example that galectin-1
displays pro-migratory effects in normal56 and cancer24 cells
in concentrations fitting with bell-shaped curves. In addition,
specific galectins are involved in the migration of specific types
of normal or cancer cells as for example galectin-1 in gliomas,24
galectin-325 and galectin-826 in colon cancer cells, and galectin-7
in normal and neoplastic pluristratified epithelia.8,16 If the
optimum concentration for an antigalectin compound observed
in a given cancer type is due to inhibition of different galectins
with opposing effects and with different affinity for the inhibitor,
then we can continue to develop highly specific compounds for
a given galectin type in order to overcome, at least partly, the
difficulty of dosing to reach an optimum in terms of antimi-
gratory activity.
Measured by a Fluorescence Polarization Assay Previously Described in
a
Detail47,48
galectin
1
3
7
8N
104 ( 43 2.2 ( 0.3
42 ( 12 1.4 ( 0.3
9N
11
16
17
18
19
6.4 ( 1.5b
4.4 ( 1.0
10.3 ( 1.5
16.8 ( 2.5
14.7 ( 6.5
1.6 ( 0.4
0.69 ( 0.19 22 ( 4.4
3.6 ( 1.5
3.8 ( 1.6
10.7 ( 2.6
22 ( 15
190 ( 43 135 ( 11 15 ( 5.8
24 ( 8.5
48 ( 4.5
24 ( 4
8.3 ( 0.8
2.8 ( 1.4
100 ( 3
Selected Diamide Reference Compounds:43
4a
35c
1.7
0.050
0.16
4.3
2.1
1.7
high
7.0
1.8
0.73
4b ≈2c
≈100
4c
2a
3a
9.6c
>100
Selected LacNAc 3′-Amide Reference Compound:40
4.4 7.6 high
Selected Lactose C2-Ester Reference Compound:41
2.5 54 530
Reference Compounds:
25c
14c
>100
1.6
41,43
1
20
21
78 ( 20d
49
59
160
550
110
61
1000
62
38
490
23
150 ( 26d
420 ( 130d 160
a The methyl ꢀ-glycosides of LacNAc (20) and lactose (21) are included
as reference compounds. b Average and standard deviation of 4-8 single
point measurements. Galectin-7 and -9N were measured at 0 °C, while
galectin-1, -3, and -8N were measured 20 °C. c Determined at 0 °C. d New
values obtained at 20 °C. Published literature values41 were obtained at 0
°C.
Chart 2. Structures of Reference C3′-Modified LacNAc
Derivative 2a, 1-Napthoate of Lactose 3a, and
Diamido-thiodigalactosides 4a-c
We recently reviewed the various antimigratory compounds
that reached clinical trials in oncology, along with the pharma-
cological tools that have been used to identify these antimigra-
tory compounds.53 Most of the antimigratory compounds have
been identified thanks to the 3D radial migration assay, as
cilengitide, or to the Boyden chamber assay, as most of the
antimetalloproteinase compounds.53 We are only few groups
to routinely use computer-assisted phase-contrast microscopy
(quantitative videomicroscopy) to identify antimigratory com-
pounds as potential nontoxic anticancer agents.53 Within our
own experience using this tool, the compounds under study,
i.e., compounds 16 and 18, display higher antimigratory effects
on cancer cells than compounds such as 3-aryl-quinolone
derivatives,50 lactosylated steroids,51 and cimetidine, an anti-
inflammatory compound.57 Combining each of these antimi-
gratory compounds with anticancer drugs exerting cytotoxic
effects though pro-apoptotic and/or pro-autophagic effects
significantly increases the therapeutic benefits contributed by
these cytotoxic drugs in a broad panel of in vivo syngeneic
mouse tumor models and human xenografts.50,51,53,57
Conclusions
explanation is that inhibition of only a small fraction of a
galectin is required to have an effect on cell migration due to
downstream amplified signaling pathways. It is also possible
that the inhibitors act more efficiently on a galectin (or other
lectin) not investigated in the binding assay described above.
The apparent discrepancies between the inhibitory effects of
compounds 16 and 18 on A549 and PC-3 cell motility can be
explained, at least partly, by two distinct features, i.e., (i)
Thiodigalactoside diesters 11 and 16-19 have been prepared
by a straightforward three-step synthesis from commercial
thiodigalactoside 1 and included at least one with improved
inhibitory potency against galectin-1, -3, -7, or -9N. Two of
the esters, the 3-methoxybenzoate 16 and the 1-naphthoate 18,
were further shown to display potent antimigratory properties
toward experimental lung and prostate cancer cells. The ease
of preparation of the diesters 16 and 18 together with their

8112 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Delaine et al.
Figure 1. The effects of compounds 16 and 18 on A549 and PC-3 cell motility have been determined in cell culture by quantitative video microscopy
for 12 h (the open boxes) or 24 h (the shaded boxes). The open and black squares represent mean values, while the open and shaded boxes represent
the standard errors of the mean. Each experiment has been carried out in triplicate. The levels of motility (calculated in µm per hour) have been
arbitrarily defined as 100% in the control conditions (no inhibitor). * ) p < 0.05; ** ) p < 0.01; *** ) p < 0.001 as compared to controls
(Mann-Whitney parametric test).
Bis-[3-O-(3-methoxybenzoyl)-ꢀ-D-galactopyranosyl]sulfane 16.
Compound 12(32 mg, 0.040 mmol) and Dowex 50XB (50 mg) were
refluxed in MeOH (3 mL) for 2 h. The mixture was filtered through
celite, concentrated, and flash chromatographed (20:1 f 15:1
CH2Cl2:EtOH) to give 16 (23 mg, 94%). δH (400 MHz, CD3OD)
3.66-3.82 (6H, m, H-5, H-6, H-6′), 3,85 (6H, s, Me), 4.03 (2H,
at, J2,3 9.8 Hz, H-2), 4.19 (2H, d, J4,3 3.2 Hz, H-4), 4.90 (2H, d,
J1,2 9.8 Hz, H-1), 5.05 (2H, dd, J3,4 3.2 Hz, J3,2 9.8 Hz H-3), 7.17
(2H, ddd, J 1.2 Hz, J 2.8 Hz, J 8.4 Hz, Ar-H), 7.39 (2H, t, J 8.0
Hz, Ar-H), 7.65 (2H, dd, J 1.2 Hz, J 2.8 Hz, Ar-H), 7.71 (2H,
adt, J 1.2 Hz, J 7.7 Hz, Ar-H). (HRMS calcd for C28H34O14SNa
(MNa+) 649.1567; found 649.1570.) In a similar manner were
prepared 17, 18, and 19.
antimigratory effects on cancer cells render them promising
leads for further development into novel antitumor drugs, for
example to combat glioblastoma, a cancer associated with dismal
prognosis.58 Glioblastoma cells are highly migratory cancer cells
that diffusely invade the brain and exhibit an innate resistance
to apoptosis.58 Decreasing migration in glioblastoma cells render
them sensitive to apoptosis.57,58 We will thus investigate in the
near future whether diesters 16 and 18, in association with
cytotoxic agents (as for example carmustine (BCNU) or
Temozolomide) contribute significant therapeutic benefits in
human orthotopic models of human glioblastoma xenotrans-
planted into the brains of immunocompromized mice.15,34,51,57
Bis-[3-O-(4-methylbenzoyl)-ꢀ-D-galactopyranosyl]sulfane 17. δH
(400 MHz, CD3OD) 2.42 (6H, s, Me), 3.67-3.84 (6H, m, H-5,
H-6, H-6′), 4.02 (2H, at, J 9.8 Hz, H-2), 4.19 (2H, d, J4,3 3.0 Hz,
H-4), 4.90 (2H, d, J1,2 9.8 Hz, H-1), 5.03 (2H, dd, J3,4 3.0 Hz, J3,2
9.8 Hz, H-3), 7.31 (4H, br d, J 7.6 Hz, Ar-H), 8.01 (4H, br d, J
8.4 Hz, Ar-H). (HRMS calcd for C28H34O12SNa (MNa+) 617.1669;
found 617.1671.)
Bis-(3-O-1-naphthoyl-ꢀ-D-galactopyranosyl)sulfane 18. δH (400
MHz, CD3OD) 3.72-3.88 (6H, m, H-5, H-6, H-6′), 4.10 (2H, at,
J 9.8 Hz, H-2), 4.31 (2H, d, J4,3 3.1 Hz, H-4), 4.96 (2H, d, J1,2 9.8
Hz, H-1), 5.17 (2H, dd, J3,4 3.1 Hz, J3,2 9.8 Hz, H-3), 7.53-7.64
(6H, m, Ar-H), 7.95 (2H, br d, J 7.6 Hz, Ar-H), 8.11 (2H, d, J
8.0 Hz, Ar-H), 8.33 (2H, dd, J 1.2 Hz, J 7.2 Hz, Ar-H), 8.93
(2H, br d, J 8.4 Hz, Ar-H). (HRMS calcd for C34H34O12SNa
(MNa+) 689.1669; found 689.1659.)
Experimental Section
Bis-(3-O-benzoyl-ꢀ-D-galactopyranosyl)sulfane 11. Compound 7
(28 mg, 0.038 mmol) was stirred with Dowex 50XB (ca. 30 mg)
in methanol (1 mL) at room temperature for 24 h. After this time,
TLC (4:1 ethyl acetate:methanol) showed the presence of a single
product (Rf 0.5). The mixture was filtered, concentrated in vacuo,
and purified by flash column chromatography (19:1 f 4:1 ethyl
acetate:methanol) to give 11 (13 mg, 61%) as a colorless oil. δH
(400 MHz, CD3OD) 3.68-3.75 (4H, m, H-5, H-6), 3.82 (2H, dd,
J6′,5 6.3 Hz, J6′,6 10.3 Hz, H-6′), 4.03 (2H, at, J 9.8 Hz, H-2), 4.20
(2H, d, J4,3 3.2 Hz, H-4), 4.90 (2H, d, J1,2 9.8 Hz, H-1), 5.05 (2H,
dd, J3,2 9.8 Hz, J3,4 3.2 Hz, H-3), 7.47-7.50 (4H, m, Ar-H),
7.59-7.63 (2H, m, Ar-H), 8.11-8.13 (4H, m, Ar-H). δC (100.6
MHz, CD3OD) 62.7 (t, C-6), 68.5 (d, C-4), 69.3 (d, C-2), 79.1 (d,
C-3), 80.7 (d, C-5), 85.3 (d, C-1), 129.5, 130.9, 134.3 (3d, Ar-
CH), 131.5 (s, Ar-C), 167.7 (s, CdO). m/z (FAB+) 589 (M +
Na+, 100%). (HRMS calcd for C26H30O12SNa (MNa+) 589.1356;
found 589.1359).
Bis-(3-O-2-naphthoyl-ꢀ-D-galactopyranosyl)sulfane 19. δH (300
MHz, CD3OD) 3.70-3.90 (6H, m, H-5, H-6, H-6′), 4.10 (2H, at,
J 9.8 Hz, H-2), 4.25 (2H, d, J4,3 3.2 Hz, H-4), 4.90 (2H, d, J1,2 9.9
Hz, H-1), 5.10 (2H, dd, J3,4 3.2 Hz, J3,2 9.8 Hz, H-3), 7.54-7.70
(4H, m, Ar-H), 7.88-7.95 (4H, m, Ar-H), 8.03 (2H, br d, J 7.8

Antimigratory Thiodigalactoside Esters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8113
(21) Liu, F.-T. Regulatory Roles of Galectins in the Immune Response.
Int. Arch. Allergy Immunol. 2005, 136, 385–400.
(22) Danguy, A.; Camby, I.; Kiss, R. Biochim. Biophys. Acta 2002, 1572,
285–293.
Hz, Ar-H), 8.12 (2H, dd, J 1.6 Hz, J 8.6 Hz, Ar-H), 8.73 (2H, br
s, Ar-H). (HRMS calcd for C34H34O12SNa (MNa+) 689.1669;
found 689.1649.)
(23) Liu, F.-T.; Rabinovich, G. A. Galectins as modulators of tumour
progression. Nat. ReV. Cancer 2005, 5, 29–41.
Acknowledgment. We thank Lund University, the Swedish
Research Council, and the programs “Glycoconjugates in
Biological Systems” (GLIBS) and “Chemistry for Life Sciences”
sponsored by the Swedish Strategic Research Foundation for
financial support. We thank Barbro Kahl-Knutsson for perform-
ing the fluorescence polarization assay. Part of this work has
been granted by the “Fonds Yvonne Boe¨l” (Brussels, Belgium).
Marie Le Mercier is the holder of a Grant-Te´le´vie (Brussels,
Belgium). Robert Kiss is a Director of Research with the “Fonds
National de la Recherche Scientifique” (FNRS, Brussels,
Belgium).
(24) Camby, I.; Belot, N.; Lefranc, F.; Sadeghi, N.; de Launoit, Y.; Kaltner,
H.; Musette, S.; Darro, F.; Dabguy, A.; Salmon, I.; Gabius, H. J.;
Kiss, R. Galectin-1 modulates human glioblastoma cell migration into
the brain through modifications to the actin cytoskeleton and levels
of expression of small GTPases. J. Neuropathol. Exp. Neurol. 2002,
61, 585–596.
(25) Hittelet, A.; Legendre, H.; Nagy, N.; Bronckart, Y.; Pector, J. C.;
Salmon, I.; Yeaton, P.; Gabius, H. J.; Kiss, R.; Camby, I. Upregulation
of galectins-1 and -3 in human colon cancer and their role in regulating
cell migration. Int. J. Cancer 2003, 103, 370–379.
(26) Nagy, N.; Bronckart, Y.; Camby, I.; Legendre, H.; Lahm, H.; Kaltner,
H.; Hadari, Y.; Van Ham, P.; Yeaton, P.; Pector, J. C.; Zick, Y.;
Salmon, I.; Danguy, A.; Kiss, R.; Gabius, H.-J. Galectin-8 expression
decreases in cancer compared with normal and dysplastic human colon
tissue and acts significantly on human colon cancer cell migration as
a suppressor. Gut 2002, 50, 392–401.
Supporting Information Available: Experimental procedures
and physical data for compounds 5-10, 12-15. 1H NMR spectra,
RP-HPLC chromatograms, and HPLC purities for compounds 11
and 16-19. This material is available free of charge via the Internet
at http://pubs.acs.org.
(27) Toscano, M. A.; Bianco, G. A.; Ilarregui, J. M.; Croci, D. O.; Correale,
J.; Hernandez, J. D.; Zwirner, N. W.; Poirer, F.; Riley, E. M.; Baum,
L.; Rabinovich, G. A. Differential glycosylation of TH1, TH2 and
TH-17 effector cells selectively regulates susceptibility to cell death.
Nat. Immun. 2007, 825–834.
References
¨
(28) Carlsson, S.; Oberg, C. T.; Carlsson, M. C.; Sundin, A.; Nilsson, U. J.;
(1) Zick, Y.; Eisenstein, M.; Goren, R. A.; Hadari, Y. R.; Levy, Y.; Ronen,
D. Role of galectin-8 as a modulator of cell adhesion and cell growth.
Glycoconjugate J. 2004, 19, 517–526.
(2) Ochieng, J.; Furtak, V.; Lukyanov, P. Extracellular functions of
galectin-3. Glycoconjugate J. 2004, 19, 527–535.
(3) Rabinovich, G. A.; Toscano, M. A.; Ilarregui, J. M.; Rubinstein, N.
Shedding light on the immunomodulatory properties of galectins:
Novel regulators of innate and adaptive immune responses. Glyco-
conjugate. J. 2004, 19, 565–573.
(4) Huflejt, M.; Leffler, H. Galectin-4 in normal tissues and cancer.
Glycoconjugate J. 2004, 20, 247–255.
(5) Takenaka, Y.; Fukumori, T.; Raz, A. Galectin-3 and metastasis.
Glycoconjugate. J. 2004, 19, 543–549.
(6) Patterson, R. J.; Wang, W.; Wang, J. L. Understanding the biochemical
activities of galectin-1 and galectin-3 in the nucleus. Glycoconjugate
J. 2004, 19, 499–506.
(7) Hsu, D. K.; Liu, F. T. Regulation of cellular homeostasis by galectins.
Glycoconjugate J. 2004, 19, 507–515.
(8) Saussez, S.; Camby, I.; Toubeau, G.; Kiss, R. Galectins as modulators
of tumor progression in head and neck squamous cell carcinomas.
Head Neck 2007, 29, 874–884.
Smith, D.; Cummings, R. D.; Almkvist, J.; Karlsson, A.; Leffler, H.
Affinity of galectin-8 and its carbohydrate recognition domains for
ligands in solution and at the cell surface. Glycobiology 2007, 17,
663–676.
(29) Carlsson, S.; Carlsson, M. C.; Leffler, H. Intracellular sorting of
galectin-8 based on carbohydrate fine specificity. Glycobiology 2007,
17, 906–912.
(30) Demetriou, M.; Granovsky, M.; Quaggin, S.; Dennis, J. W. Negative
regulation of T-cell activation and autoimmunity by Mgat5 N-
glycosylation. Nature 2001, 409, 733–739.
(31) Partridge, E. A.; Le Roy, C.; Di Guglielmo, G. M.; Pawling, J.;
Cheung, P.; Granovsky, M.; Nabi, I. R.; Wrana, J. L.; Dennis, J. W.
Regulation of cytokine receptors by Golgi N-glycan processing and
endocytosis. Science 2004, 306, 120–124.
(32) Lau, K. S.; Partridge, E. A.; Grigorian, A.; Silvescu, C. I.; Reinhold,
V. N.; Demetriou, M.; Dennis, J. W. Complex N-glycan number and
degree of branching cooperate to regulate cell proliferation and
differentiation. Cell 2007, 129, 123–134.
(33) Mathieu, V.; Le Mercier, M.; De Neve, N.; Sauvage, S.; Gras, T.;
Roland, I.; Lefranc, F.; Kiss, R. Galectin-1 knockdown increases
sensitivity to Temozolomide in a B16F10 mouse metastatic melanoma
model. J. InVest. Dermatol. 2007, 127, 2399–2410.
(9) Leffler, H.; Carlsson, S.; Hedlund, M.; Qian, Y. Introduction to
galectins. Glycoconjugate J. 2004, 19, 433–440.
(10) Dumic, J.; Dabelic, S.; Flo¨gel, M. Galectin-3: An open-ended story.
Biochim. Biophys. Acta 2006, 1760, 616–635.
(34) Le Mercier, M.; Lefranc, F.; Mijatovic, T.; Debeir, O.; Haibe-Kains,
B.; Bontempi, G.; Decaestecker, C.; Kiss, R.; Mathieu, V. Evidence
of galectin-1 involvement in glioma chemoresistance. Toxicol. Appl.
Pharmacol. 2008, 229, 172–183.
(35) Fukumori, T.; Kanayama, H.-o.; Raz, A. The role of galectin-3 in
cancer drug resistance. Drug Resist. Update 2007, 10, 101–108.
(36) Pieters, R. J. Inhibition and Detection of Galectins. ChemBioChem
2006, 7, 721–728.
(37) Ingrassia, L.; Camby, I.; Lefranc, F.; Mathieu, V.; Nshimyumukiza,
P.; Darro, F.; Kiss, R. Anti-galectin compounds as potential anti-cancer
drugs. Curr. Med. Chem. 2006, 13, 3513–3527.
(38) Bianchet, M. A.; Ahmed, H.; Vasta, G. R.; Amzel, L. M. Soluble
beta-galactosyl-binding lectin (galectin) from toad ovary: crystal-
lographic studies of two protein-sugar complexes. Proteins 2000, 40,
378–388.
(39) So¨rme, P.; Qian, Y.; Nyholm, P.-G.; Leffler, H.; Nilsson, U. J. Low
micromolar inhibitors of galectin-3 based on 3′-derivatization of
N-acetyllactosamine. ChemBioChem 2002, 3, 183–189.
(40) So¨rme, P.; Arnoux, P.; Kahl-Knutsson, B.; Leffler, H.; Rini, J. M.;
Nilsson, U. J. Structural and thermodynamic studies on cation-Π
interactions in lectin-ligand complexes: High-affinity galectin-3
inhibitors through fine-tuning of an arginine-arene interaction. J. Am.
Chem. Soc. 2005, 127, 1737–1743.
(11) Camby, I.; Le Mercier, M.; Lefranc, F.; Kiss, R. Galectin-1: a small
protein with major functions. Glycobiology 2006, 16, 137R–157R.
(12) Perillo, N. L.; Pace, K. E.; Seilhamer, J. J.; Baum, L. G. Apoptosis of
T cells mediated by galectin-1. Nature 1995, 378, 736–739.
(13) Nakahara, S.; Oka, N.; Raz, A. On the role of galectin-3 in cancer
apoptosis. Apoptosis 2005, 10, 267–275.
(14) Delacour, D.; Greb, C.; Koch, A.; Salomonsson, E.; Leffler, H.; Le
Bivic, A.; Jacob, R. Apical Sorting by Galectin-3-Dependent Glyco-
protein Clustering. Traffic 2007, 8, 379–388.
(15) Le Mercier, M.; Mathieu, V.; Haibe-Kains, B.; Bontempi, G.;
Mijatovic, T.; Decaestecker, C.; Kiss, R.; Lefranc, F. Knocking down
galectin-1 in human Hs683 glioblastoma cells impairs both angiogen-
esis and endoplasmic reticulum stress responses. J. Neuropathol. Exp.
Neurol. 2008, 67, 456–469.
(16) Saussez, S.; Kiss, R. Galectin-7. Cell. Mol. Life Sci. 2006, 63, 686–
697.
(17) Hirashima, M.; Kashio, Y.; Nishi, N.; Yamauchi, A.; Imaizumi, T. A.;
Kageshita, T.; Saita, N.; Nakamura, T. Galectin-9 in physiological
and pathological conditions. Glycoconjugate. J. 2004, 19, 593–600.
(18) Sato, S.; Nieminen, J. Seeing strangers or announcing “danger”:
Galectin-3 in two models of innate immunity. Glycoconjugate. J. 2004,
19, 583–591.
(41) Cumpstey, I.; Salomonsson, E.; Sundin, A.; Leffler, H.; Nilsson, U. J.
Studies of Arginine-Arene Interactions through Synthesis and Evalu-
ation of a Series of Galectin-Binding Aromatic Lactose Esters.
ChemBioChem 2007, 8, 1389–1398.
(19) Rubinstein, N.; Toscano, M. A.; Ilarrgui, J. M.; Bianco, G. A.;
Rabinovich, G. A. The Sweet Kiss of Death: A Link between Galectin-
1, Glycosylation and the Generation of Immune Privilege. Trends
Glycosci. Glycotechnol. 2005, 17, 133–143.
(20) Ilarregui, J. M.; Bianco, G. A.; Toscano, M. A.; Rabinovich, G. A.
The coming of age of galectins as immunomodulatory agents: impact
of these carbohydrate binding proteins in T cell physiology and chronic
inflammatory disorder. Ann. Rheum. Dis. 2005, 64, iv96-iv103.
(42) Cumpstey, I.; Sundin, A.; Leffler, H.; Nilsson, U. J. C2-Symmetrical
thiodigalactoside bis-benzamido derivatives as high-affinity inhibitors
of galectin-3: Efficient lectin inhibition through double arginine-arene
interactions. Angew. Chem., Int. Ed. 2005, 44, 5110–5112.

8114 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Delaine et al.
(43) Cumpstey, I.; Salomonsson, E.; Sundin, A.; Leffler, H.; Nilsson, U. J.
Double affinity amplification of galectin-ligand interactions through
arginine-arene interactions: Synthetic, thermodynamic, and compu-
tational studies with aromatic diamido-thiodigalactosides. Chem.sEur.
J. 2008, 14, 4233–4245.
(44) Bogusiak, J.; Szeja, W. Synthesis of bis(2,3,4,6-tetra-O-acetyl-ꢀ-D-
glucopyranosyl and -galactopyranosyl) sulfide using phase-transfer
catalysis. Carbohydr. Res. 1985, 141, 165–167.
(45) Gangadharmath, U. B.; Demchenko, A. V. Nickel(II) Chloride-
Mediated Regioselective Benzylation and Benzoylation of Diequatorial
Vicinal Diols. Synlett 2004, 2191–2193.
(46) Pelyva´s, I. F.; Lindhorst, T. K.; Streicher, H.; Thiem, J. Regioselective
Acylation of Carbohydrates with 1-Acyloxy-1H-benzotriazoles. Syn-
thesis 1991, 1015–1018.
mouse lymphoma and human glioblastoma. J. Med. Chem. 2006, 49,
1800–1807.
(52) Huang, L. J.; Chen, S. X.; Huang, Y.; Luo, W. J.; Jiang, H. H.; Hu,
Q. H.; Zhang, P. F.; Yi, H. Proteomics-based identification of secreted
protein dihydrodiol dehydrogenase as a novel serum markers of non-
small cell lung cancer. Lung Cancer 2006, 54, 87–94.
(53) Decaestecker, C.; Debeir, O.; Van Ham, P.; Kiss, R. A review of 2D
and 3D assays for the quantitative analysis of cell migration. Med.
Res. ReV. 2007, 27, 149–176.
(54) Lefranc, F.; Camby, I.; Belot, N.; Bruyneel, E.; Chaboteaux, C.;
Brotchi, J.; Mareel, M.; Salmon, I.; Kiss, R. Gastrin significantly
modifies the migratory abilities of experimental glioma cells. Lab.
InVest. 2002, 82, 1241–1252.
(55) Mijatovic, T.; Gailly, P.; Mathieu, V.; De Neve, N.; Yeaton, P.; Kiss,
R.; Decaestecker, C. Neurotensin is a versatile modulator of in vitro
human pancreatic ductal adenocarcinomas cell (PDAC) migration. Cell
Oncol. 2007, 29, 315–326.
(56) Delbrouck, C.; Doyen, I.; Belot, N.; Decaestecker, C.; Ghanooni, R.;
de Lavareille, A.; Kaltner, H.; Choufani, G.; Danguy, A.; Vanden-
hoven, G.; Gabius, H.-J.; Hassid, S.; Kiss, R. Galectin-1 is overex-
pressed in nasal polyps under budesonide and inhibits eosinophil
migration. Lab. InVest. 2002, 82, 147–158.
(57) Lefranc, F.; James, S.; Camby, I.; Gaussin, J. F.; Darro, F.; Brotchi,
J.; Gabius, H.-J.; Kiss, R. Combined cimetidine and Temozolomide,
compared with Temozolomide alone: Significant increases in survival
in nude mice bearing U373 human glioblastoma multiforme orthotopic
xenografts. J. Neurosurg. 2005, 102, 706–714.
(47) So¨rme, P.; Kahl-Knutsson, B.; Huflejt, M.; Nilsson, U. J.; Leffler, H.
Fluorescence polarization as an analytical tool to evaluate galectin-
ligand interactions. Anal. Biochem. 2004, 334, 36–47.
(48) Cumpstey, I.; Carlsson, S.; Leffler, H.; Nilsson, U. J. Synthesis of a
phenyl thio-ꢀ-D-galactopyranoside library from 1,5-difluoro-2,4-dini-
trobenzene: discovery of efficient and selective monosaccharide
inhibitors of galectin-7. Org. Biomol. Chem. 2005, 3, 1922–1932.
(49) Debeir, O.; Van Ham, P.; Kiss, R.; Decaestecker, C. Tracking of
Migrating Cells Under Phase-Contrast Video Microscopy With
Combined Mean-Shift Processes. IEEE Trans. Med. Imaging 2005,
24, 697–711.
(50) Joseph, B.; Darro, F.; Behard, A.; Lesur, B.; Collignon, F.; Decae-
stecker, C.; Frydman, A.; Guillaumet, G.; Kiss, R. 3-Aryl-quinolone
derivatives: Synthesis and characterization of in vitro and in vivo
antitumor effects with emphasis on a new therapeutical target
connected with cell migration. J. Med. Chem. 2002, 45, 2543–2555.
(51) Ingrassia, L.; Nshimyumukiza, P.; Dewelle, J.; Lefranc, F.; Wlodarc-
zak, L.; Thomas, S.; Dielie, G.; Chiron, C.; Zedde, C.; Tisnes, P.; van
Soest, R.; Braekman, J. J.; Darro, F.; Kiss, R. A lactosylated steroid
contributes in vivo therapeutic benefits in experimental models of
(58) Lefranc, F.; Brotchi, J.; Kiss, R. Possible future issues in the treatment
of glioblastomas: Special emphasis on cell migration and the resistance
of migrating glioblastoma cells to apoptosis. J. Clin. Oncol. 2005,
23, 2411–2422.
JM801077J