A lactosylated steroid contributes in vivo therapeutic benefits in experimental models of mouse lymphoma and human glioblastoma

Article abstract of DOI:10.1021/jm050971v

Various mono- and disaccharides were grafted onto a steroid backbone. Whereas in vitro these glycosylated steroids had no cytotoxic effects on six different human cancer cell lines, several of the glycosylated steroids under study did significantly modify the levels of in vitro migration of the human U373 glioblastoma, the A549 non-small-cell-lung cancer (NSCLC), and the PC-3 prostate cancer cells, with more pronounced effects in the case of a monosubstituted β-L-fucopyranosyl-steroid (19), a monosubstituted β-D-isomaltosyl-steroid (22), and a monosubstituted β-D-lactosyl- steroid (24). These three compounds significantly increased the survival of conventional mice grafted subcutaneously with the P388 lymphoma, a lymphoma that metastasizes toward the liver. In vivo, the monosubstituted β-D-lactosyl-steroid (24) also increased the antitumor effectiveness of cisplatin, a cytotoxic pro-apoptotic drug, in the case of the P388 lymphoma model. This compound also increased the survival of immunodeficient mice into whose brains human U373 glioblastoma cells had been orthotopically grafted.

Full text of DOI:10.1021/jm050971v

1800
J. Med. Chem. 2006, 49, 1800-1807
A Lactosylated Steroid Contributes in Vivo Therapeutic Benefits in Experimental Models of
Mouse Lymphoma and Human Glioblastoma
Laurent Ingrassia,† Prosper Nshimyumukiza,† Janique Dewelle,† Florence Lefranc,‡ Lise Wlodarczak,† Ste´phanie Thomas,†
Gwenae¨l Dielie,† Christelle Chiron,§ Chantal Zedde,§ Pierre Tisne`s,§ Rob van Soest,| Jean-Claude Braekman,
Francis Darro,† and Robert Kiss*,#,∇
Unibioscreen SA, 40 AVenue Joseph Wybran 1070 Brussels, Belgium, SerVice de Neurochirurgie, Hoˆpital Erasme, UniVersite´ Libre de
Bruxelles, Brussels, Belgium, Laboratoire de Synthe`se et Physico-Chimie de Mole´cules d’Inte´reˆt Biologique, Unite´ Mixte de Recherche No.
5068sCentre National de la Recherche Scientifique, UniVersite´ Paul Sabatier, Toulouse, France, Head Curator Section InVertebrates,
Zoologish Museum, UniVersity of Amsterdam, Amsterdam, The Netherlands, SerVice de Chimie Organique, Faculte´ des Sciences, UniVersite´
Libre de Bruxelles, Brussels, Belgium, and Laboratoire de Toxicologie, Institut de Pharmacie, UniVersite´ Libre de Bruxelles, Brussels, Belgium
ReceiVed September 29, 2005
Various mono- and disaccharides were grafted onto a steroid backbone. Whereas in vitro these glycosylated
steroids had no cytotoxic effects on six different human cancer cell lines, several of the glycosylated steroids
under study did significantly modify the levels of in vitro migration of the human U373 glioblastoma, the
A549 non-small-cell-lung cancer (NSCLC), and the PC-3 prostate cancer cells, with more pronounced effects
in the case of a monosubstituted â-L-fucopyranosyl-steroid (19), a monosubstituted â-D-isomaltosyl-steroid
(22), and a monosubstituted â-D-lactosyl-steroid (24). These three compounds significantly increased the
survival of conventional mice grafted subcutaneously with the P388 lymphoma, a lymphoma that metastasizes
toward the liver. In vivo, the monosubstituted â-D-lactosyl-steroid (24) also increased the antitumor
effectiveness of cisplatin, a cytotoxic pro-apoptotic drug, in the case of the P388 lymphoma model. This
compound also increased the survival of immunodeficient mice into whose brains human U373 glioblastoma
cells had been orthotopically grafted.
Introduction
patients) in the case of the MXT mouse mammary adenocar-
cinoma.5 In the same way, we have shown in experimental
glioblastomas that cimetidine (a H2 receptor antagonist6)
increases the therapeutic effects of the pro-autophagic drug
temozolomide by decreasing the levels of migration of malignant
glial cells.7
One of four individuals dies from cancer in industrialized
countries, and 90% of cancer patients die from their metastases.
Once a metastatic cancer has been diagnosed in a patient, the
probability that the patient will survive for more than a year
after the initial diagnosis of his/her cancer is less than 50%.
While glioblastomas do not metastasize outside the brain, they
do remain associated with the most dismal prognosis for any
type of human cancer because they diffusely invade the brain
parenchyma during their development and, in so doing, avoid
total surgical resection. Migrating cancer cells are protected
against apoptosis.1-4 Compounds that are able to reduce the
migration levels of apoptotic-resistant cancer cells not only delay
both the formation of metastases by cancer cells emerging from
primary tumor sites and the invasion of the brain parenchyma
by glioblastoma cells but also restore a certain level of sensitivity
to apoptosis in these slowly migrating cells.1-4 We have already
shown that, in vitro, nontoxic 2-quinolone derivatives have an
antimigratory effect on cancer cells and are a cause of additive
in vivo benefits when combined with etoposide or adriamycin
(two pro-apoptotic and cytotoxic compounds used to treat cancer
In our quest to identify novel anticancer drugs with a potential
antimigratory effect we decided to perform the bioguided
fractionation of various marine sponges because these organisms
are known to defend themselves against invaders by secreting
cytotoxic and/or antimigratory compounds.8,9 We observed that
a number of compounds with steroid backbones from different
sponges display both weak antimigratory and weak cytotoxic
effects (unpublished data). We decided to start from a related
and more accessible steroid skeleton (compound 1, Scheme 1)
in order to become independent of marine sponge sourcing.
Derivatives of compound 1 that we have synthesized as detailed
elsewhere10 displayed some cytotoxic effects when added
repeatedly to the human cancer cell culture media.11,12 Antimi-
gratory compounds that could be used to successfully combat
migrating cancer cells have to be administered chronically to
cancer patients. These compounds must therefore be noncyto-
toxic. We derived a series of novel steroids, of which compound
4 (see Scheme 1) was associated with a total absence of
cytotoxicity and a certain level of antimigratory activity.11,12
We then chose to synthesize novel glycosylated derivatives from
compound 4 in order to endow this type of compound with real
antimigratory, but noncytotoxic activity. The data obtained with
the novel compounds that we describe in the present work show
that we placed special emphasis on fucosylated and lactosylated
analogues because (i) fucosylated moieties are known to be able
to modulate tumor cell biology, especially in glioblastomas,13,14
and (ii) â-lactoside moieties can bind to the galectins involved
in the formation of metastases from epithelial cancers (carci-
* Author to whom correspondence should be addressed: Laboratory of
Toxicology, Institute of Pharmacy, Universite´ Libre de Bruxelles, Campus
de la Plaine, CP205/1, Boulevard du Triomphe, 1050 Brussels, Belgium.
Phone: +32 477 62 20 83. Fax: +322 332 53 35. E-mail: rkiss@ulb.ac.be.
† Unibioscreen SA.
‡ Service de Neurochirurgie, Hoˆpital Erasme, Universite´ Libre de
Bruxelles.
§ Universite´ Paul Sabatier.
| University of Amsterdam.
Service de Chimie Organique, Faculte´ des Sciences, Universite´ Libre
de Bruxelles.
# Laboratoire de Toxicologie, Institut de Pharmacie, Universite´ Libre de
Bruxelles.
3 A Director of Research with the Belgian National Fund for Scientific
Research (FNRS, Belgium).
10.1021/jm050971v CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/09/2006

In ViVo Therapeutic Benefits from a Lactosylated Steroid
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1801
Scheme 1
(a) K2CO3, H2O, MeOH; (b) tert-butyldiphenylsilylchloride, imidazole, DMF; (c) 1-bromo-2,5-dimethoxybenzene, Mg, 1,2-dibromoethane, CuI, Et2O;
(d) FNtBu4, THF.
Table 1. (A) Compounds Synthesized from 4 and (B) Compounds
Synthesized from 5
Scheme 2
(a) NaBH4, MeOH.
Chemistry
The steroid derivative 4 was prepared in four steps (Scheme
1). The intermediate 2 can be synthesized in four steps with an
overall yield of 27%, as described previously.17 We developed
here a new method enabling us to obtain compound 2 in fewer
steps with a higher overall yield (Scheme 1), as detailed in the
Experimental Section.
The synthesized compounds under study are summarized in
Tables 1A and 1B.
Pharmacology
In Vitro Cytotoxic Activity and in Vivo Tolerance. None
of the glycosylated steroids under study had any significant
cytotoxic effect on six different human cancer cell lines
(including the Hs683 and U373 glioblastoma, the HCT-15 colon,
the PC-3 prostate, the A549 NSCLC, and the MCF-7 breast
cancer models). Indeed, the IC50 values of each of these
compounds were higher than 10 µM for each of the six cancer
cell lines under study. The IC50 value represents the concentra-
tion that reduces by 50% the overall growth level of a cell
population after 3 days in the presence of the drug. In the same
way, all these compounds were tolerated very well in vivo
because they induced no toxic effects, at least in terms of both
weight development and the survival of healthy mice for a
minimum period of observation of 28 days after a single ip
administration of 160 mg/kg.
Characterization of in Vitro Antimigratory Effects. Four-
teen synthesized glycosylated steroids were submitted to in vitro
cell migration analyses on three human cancer models in the
shape of the A549 NSCLC, the PC-3 prostate cancer, and the
U373 glioblastoma models, i.e., three of the six human cell lines
on which the absence of cytotoxicity of these compounds had
previously been demonstrated. Of the 14 compounds submitted
to these cell migration analyses, only 7 had any significant (p
< 0.01) antimigratory effect on at least one tumor cell line over
nomas) and the development of malignancy in glioblastomas.15,16
We in fact hypothesized that these glycosylated steroids could
act as more or less specific antagonists for the cell adhesion
receptors involved in cancer cell migration and the formation
of metastases. We used the C-3 position of compound 4 to graft
various oligosaccharide moieties (Table 1A). In addition, we
used position C-20 to develop diglycosylated analogues from
5 (Scheme 2; Table 1B) in order to determine whether the
grafting of two oligosaccharide moieties would enhance the
antimigratory activity of monoglycosylated compounds.

1802 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Ingrassia et al.
Table 2. Determination of the in Vitro Antimigratory Effects of Various Glycosylated Steroids on the Human A549 Non-Small-Cell-Lung, the PC-3
Prostate, and the U373 Glioblastoma Cancer Cell Linesa
no. of cell lines
on which a sustained
(22 h) and significant
(p < 0.01) antimigratory
effect was obsd
cancer cell lines
PC-3
A549
U373
compd
12 h
22 h
12 h
22 h
12 h
22 h
4
16
17
18
19
20
21
22
23
24
25
27
28
29
30
0 ( 5
2 ( 7
6 ( 5
9 ( 6
5 ( 9
2 ( 8
10 ( 11
6 ( 16
2 ( 10
-6 ( 5
2 ( 5
7 ( 13
-8 ( 12
-4 ( 7
0
0
0
0
1
1
0
2
0
2
1
1
0
0
0
20 ( 6**
2 ( 4
1 ( 8
2 ( 6
-1 ( 15
-8 ( 10
-15 ( 6*
-7 ( 9
4 ( 4
7 ( 4
8 ( 6
1 ( 19
-8 ( 5
-30 ( 5***
0 ( 7
-8 ( 7
-34 ( 5***
-4 ( 5
-40 ( 3***
5 ( 4
-23 ( 4***
13 ( 10
11 ( 5*
-7 ( 9
-5 ( 5
-51 ( 3***
11 ( 10
-41 ( 5***
9 ( 8
-25 ( 6**
8 ( 7
-38 ( 5***
-9 ( 9
-1 ( 9
0 ( 7
-1 ( 4
-34 ( 3***
10 ( 6
-9 ( 6
-18 ( 4**
2 ( 6
-18 ( 5**
-8 ( 9
-5 ( 5
8 ( 4
-5 ( 5
-39 ( 3***
-4 ( 4
3 ( 9
0 ( 6
-12 ( 5
-34 ( 6***
12 ( 7
-29 ( 4**
-17 ( 4*
-4 ( 6
-8 ( 4
-14 ( 4*
-12 ( 5*
-31 ( 5**
-3 ( 6
-39 ( 2***
-6 ( 4
4 ( 8
-45 ( 6***
-17 ( 6*
10 ( 4*
-4 ( 4
4 ( 7
9 ( 5
8 ( 3*
14 ( 8
-9 ( 7
-26 ( 5**
15 ( 6*
-13 ( 5
a The levels of migration were determined by means of computer-assisted videomicroscopy, with each of the compounds being tested for either 12 or 22
h in the presence (or absence for control) of a 100 nM concentration of each of the compounds under study. The control condition was arbitrarily normalized
to “0%”. The data are presented as mean values ( their standard errors. The statistical levels of significance are as follows: (*) p < 0.05; (**) p < 0.01;
(***) p < 0.001.
at least 12 h of contact (Table 2). Of these 7 compounds, only
2 had a sustained (for at least 22 h) and significant (p < 0.01)
antimigratory effect on at least 2 of the 3 tumor cell lines under
study (Table 2). These two compounds were the monosubstituted
â-D-isomaltosyl-steroid (22) and the monosubstituted â-D-
lactosyl-steroid (24) (Table 2). The monosubstituted â-L-
fucopyranosyl-steroid (19) gave rise to sustained antimigratory
activity on the U373 glioblastoma model only (Table 2).
The bisubstituted â-D-isomaltosyl-steroid (29) and the bisub-
stituted â-D-lactosyl-steroid (30) displayed a lower level of
antimigratory activity than their monosubstituted analogues
(Table 2).
In Vivo Characterization of the Antitumor Effects of
Compounds 19, 22, and 24 on a Subcutaneous Mouse
Lymphoma Model Metastasizing to the Liver. When leuke-
mic P388 cells of lymphoblastic origin18 are grafted subcutane-
ously instead of intraperitoneally, they develop as anaplastic
lymphomas.19 The P388 lymphoma is very aggressive: its cells
easily and rapidly cross the muscle wall of the peritoneal cavity
and make enormous metastatic progress in the direction of the
liver as early as one week post-tumor graft.19
As revealed by the in vitro cell migration analyses (Table
2), the two compounds under study associated with the greatest
antimigratory activity were the monosubstituted â-D-isomaltosyl-
steroid (22) and the monosubstituted â-D-lactosyl-steroid (24)
(Table 1A). We therefore assayed these two compounds in vivo
on the highly metastatic P388 lymphoma syngeneic model.
Although the monosubstituted â-L-fucopyranosyl-steroid (19)
displayed a lower level of antimigratory effects in vitro than
the other two compounds (Table 2), we nevertheless decided
to test this compound in the P388 lymphoma model as well
because in vitro data cannot easily be extrapolated in vivo.20
Figure 1A shows that while the monosubstituted â-D-isomal-
tosyl-steroid (22) and the monosubstituted â-L-fucopyranosyl-
steroid (19) had no statistically significant (p > 0.05) effect on
the growth of the subcutaneous P388 lymphoma, these two
compounds were nevertheless the cause of significant (p ) 0.04)
increases in the survival of the P388 lymphoma-bearing mice.
These antitumor effects could be considered as modest at first
glance. It should nevertheless be emphasized that the antitumor
effects associated with compounds 19 and 22 as observed here
were more pronounced than the antitumor effects observed on
the P388 lymphoma model with anticancer drugs widely used
to treat cancer patients as, for example, adriamycin, vinblastin,
vincristin, and paclitaxel.19 The possibility remains that the
increase in the survival periods brought about by the two
glycosylated steroids relates to a decrease in the metastatic
process of the subcutaneous P388 lymphoma cells toward the
liver. We are now carrying out histological analyses to
investigate this hypothesis. The P388 lymphoma is sensitive to
platinum derivatives such as cisplatin,19 a pro-apoptotic drug
that is used to treat a broad panel of human malignancies.21
The sensitivity of the P388 lymphoma to cisplatin is clearly
evidenced in Figure 1B. As observed for the monosubstituted
â-D-isomaltosyl-steroid (22) and the monosubstituted â-L-
fucopyranosyl-steroid (19) (Figure 1A), the monosubstituted
â-D-lactosyl-steroid (24) did not significantly (p > 0.05)
decrease the growth of the subcutaneous P388 lymphoma
(Figure 1B). However, as was also observed in the case of the
monosubstituted â-D-isomaltosyl-steroid (22) and the mono-
substituted â-L-fucopyranosyl-steroid (19) (Figure 1A), the
monosubstituted â-D-lactosyl-steroid (24) also significantly
increased the survival of the P388 lymphoma-bearing mice
(Figure 1B). While the effects of cisplatin on the growth of the
subcutaneous P388 lymphoma reached significant levels of
statistical significance (p < 0.05 to p < 0.01 at the various days
post-tumor graft), the increased survival rate of the P388
lymphoma-bearing mice brought about by cisplatin was no
higher than the benefit brought about by the monosubstituted
â-D-lactosyl-steroid (24) (Figure 1B). As for the monosubstituted
â-D-isomaltosyl-steroid (22) and the monosubstituted â-L-
fucopyranosyl-steroid (19), the possibility remains that the
monosubstituted â-D-lactosyl-steroid (24) decreased the meta-
static progress of the subcutaneous P388 lymphoma cells toward
the liver, while cisplatin is known to have a number of direct
cytotoxic effects on the growth of primary tumors.19,21 When
both cisplatin and the monosubstituted â-D-lactosyl-steroid (24)
were used concomitantly to treat the mice bearing the P388
lymphoma, an additional therapeutical benefit was observed with
respect to the growth of the primary subcutaneous P388
lymphoma and the survival level of the P388 lymphoma-bearing
mice (Figure 1B). Such additional effects were not observed

In ViVo Therapeutic Benefits from a Lactosylated Steroid
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1803
Figure 1. (A) The in vivo influence of a monosubstituted â-L-fucopyranosyl-steroid (19) and a monosubstituted â-D-isomaltosyl-steroid (22) on
the growth (determined as the tumor area on the Y axis) of a subcutaneous P388 lymphoma metastasizing to the liver. The mice were grafted with
the P388 lymphoma on day 0 (X axis) and were then treated (with intraperitoneal (ip) injections) or left untreated (control, with saline ip injections)
with compounds 19 or 22 five times a week (from Monday to Friday) for three consecutive weeks, with the treatment starting at the 5th day
post-tumor graft. The two compounds were administered at 40 mg/kg for each of the 15 ip injections. The T/C index represents the benefit over
control contributed by the product in terms of the percentage increases in the median mouse survival period in the treated [T] group as compared
to the median mouse survival period in the control [C] group. There were nine mice in each experimental group. (B) The in vivo influence on the
P388 mouse lymphoma model of a monosubstituted â-D-lactosyl-steroid (24) in the presence or the absence of the pro-apoptotic cisplatin cytotoxic
drug. The legend is identical to that for Figure 1A. Cisplatin was assayed ip at 10 mg/kg three times a week (Mondays, Wednesdays, and Fridays)
for three consecutive weeks, with the treatment starting at the 5th day post-tumor graft.
when either the monosubstituted â-D-isomaltosyl-steroid (22)
or the monosubstituted â-L-fucopyranosyl-steroid (19) was
added to the cisplatin (data not shown).
of the epithelial tissue (carcinomas), of the immune system
(lymphomas), or of the soft tissue (sarcomas). Cancer cell
migration also associates glioblastomas with very dismal
prognoses because of their diffuse invasion of the brain
parenchyma.1,6,7 Migrating cancer cells are resistant to apoptosis
because a number of anti-apoptotic signaling pathways are
constitutively activated in these cells, which are therefore
resistant to a large majority of currently used anticancer drugs
because these drugs are pro-apoptotic agents.1-4 New types of
drugs are needed to combat the formation of metastasis from
carcinomas or the dispersion of cancer cells into one or another
organ such as glioblastoma cells into the brain.1-7 Antimigratory
drugs assume the role of potential antimetastatic and/or anti-
invasive compounds because, in addition to reducing the
locomotive abilities of migrating cancer cells, antimigratory
compounds can also restore a certain level of sensitivity to pro-
apoptotic drugs in the case of these restricted-migratory cells.1-7
In Vivo Characterization of the Antitumor Effects of
Compound 24 on a Human Glioblastoma Xenograft Ortho-
topically Implanted into the Brains of Immunodeficient
Mice. Previous data from our group have demonstrated the
direct imvolvement of galectins in the progression of malignancy
in human glioblastomas.22-24 The fact that the monosubstituted
â-D-lactosyl-steroid (24) could at least theoretically antagonize
the biological functions of galectins in involvement in cancer
cell migration therefore prompted us to investigate whether
compound 24 could modify the survival of nude mice with
orthotopic human U373 glioblastomas in their brains. The data
in Figure 2 clearly show that compound 24 significantly
increased the survival of these mice.
Discussion
Cell migration is a highly coordinated process involving cell
adhesion, cell motility, and cell invasion. In the present study
we focused our attention on cell adhesion, i.e., the interactions
between cell surface receptors and extracellular matrix (ECM)
Cancer cell migration lies behind tumor cell dispersal to points
far distant from the primary tumor bulk and is therefore
responsible for the formation of metastases in the case of cancers

1804 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Ingrassia et al.
in the carbohydrate-recognition domain of galectins. These
subsites recognize monosaccharide units and are responsible for
the affinity and specificity of galectins to ligands.34,35 The
defined orientations of the monosaccharide units are very
important34,35 for both the recognition and a good level of
affinity of ligands to the CRD. Thus, the conformation of the
ligands36 can either enhance or eliminate the carbohydrate
recognition process by the CRD of the galectins. In natural
saccharides that bind to galectins, the galactose moiety is
typically (1-4)-linked to glucose or N-acetylglucosamine or (1-
3)-linked to N-acetylglucosamine or N-acetylgalactosamine.
These natural saccharides cannot be designated as galectin
inhibitors because they are difficult to synthesize, sensitive to
hydrolysis, and too polar to be used as drugs. To circumvent
these disadvantages Nilsson et al.37-39 replaced the glucose
moiety of the natural saccharides by less complex organic
structures. Among the galactosyl oxime ethers37 synthesized,
an indole derivative was the best inhibitor with a good level of
affinity. Other derivatives modified on anomeric carbon have
since been synthesized38,39 and have shown an affinity to
galectins. In particular, the 1-phenyl-thio derivatives of galactose
have a good level of affinity to galectin-7.38 Galectin-7 is a
potent modulator of lymphoma development25-27 and also has
a number of marked biological effects on colorectal cancers.40
Nilsson et al.39,41 also modified position 3 of the galactose
moiety because the X-ray crystal structures42 of the carbohydrate
recognition domain of human galectin-3 with N-acetyllac-
tosamine (LacNAc) showed extended binding sites close to the
3-OH function of the LacNAc. Several derivatives including
3-(1H-[1,2,3]-triazol-1-yl)-1-thio-galactoside39 and 3,3′-bis-ben-
zamido-thiodigalactosides41 are inhibitors of galectin-3, which
has a dramatic influence on both the development of me-
tastases15,43 and the resistance of carcinomas to apoptosis.44
Figure 2. Illustration of the effects of compound 24 (a monosubstituted
â-D-lactosyl-steroid) on the survival of immunodeficient mice with
human U373 glioblastoma cells grafted orthotopically into their brains.
Compound 24 (open dots) was administered iv at 40 mg/kg fifteen
times (three times a week on Mondays, Wednesdays, and Fridays) for
five consecutive weeks (with the treatment starting at the 14th day post-
tumor graft), with the control mice (black dots) receiving 15 iv injections
of saline.
components. While integrins employ protein-protein interac-
tions with ECM components, a number of other proteins
(including, for example, galectins, and selectins) use protein-
carbohydrate interactions between themselves and ECM gly-
coproteins, with the core of carbohydrate ligands for galectins
being represented by LacNAc moieties, i.e., Lewis antigens
without fucosylation, and Lewis antigens alone in the case of
selectins.1,7 The present study shows that, of the 14 glycosylated
steroids that we synthesized, the â-D-isomaltosyl-steroid and
the â-D-lactosyl-steroid had the most marked effects on cancer
cell migration in vitro. The endogenous cell surface receptors
for the â-D-isomaltosyl-steroid have not been identified so far.
In contrast, galectins can act as endogenous cell surface receptors
for â-D-lactosyl-steroids. It has already been proved that
galectins play a number of clearly defined roles in the develop-
ment of malignancy and/or cell migration in gliomas22-24 and
lymphomas.25-27 The present study clearly shows that the in
vivo combination of a â-D-lactosyl-steroid (compound 24) and
the pro-apoptotic drug cisplatin21 in an experimental metastasiz-
ing lymphoma gave rise to significant therapeutic benefits in
addition to those deriving from cisplatin alone. Compound 24
also significantly increased the survival of immunodeficient mice
bearing orthotopic xenografts of human U373 glioblastoma to
their brains. The extent of the increase in survival brought about
by compound 24 is of the same magnitude as temozolomide in
the U373 orthotopic xenograft model.28 Temozolomide is a pro-
autophagic drug29 currently associated with the a maximum level
of efficiency in the treatment of glioblastoma patients.30 We
are synthesizing novel anti-galectin derivatives from 24, and
we plan to submit them to SAR studies. Indeed, the data from
the present study reveal that a bisubstituted â-D-lactosyl-steroid
(30) displayed a lower level of antimigratory activity than its
monosubstituted analogue (24). Galectins are carbohydrate-
binding proteins containing a C-terminal carbohydrate-recogni-
tion domain (CRD). Whereas the CRDs of all known galectins
enjoy close structural homology,31 there are subtle differences
in binding affinities between specific galectins and lactosylated
moieties.32,33 The question of the specificity of galectins for
saccharide ligands involves the investigation of the binding
characteristics of this protein. Four subsites are described34,35
Interactions between fucose moieties present in glioma ECM
and cell adhesion molecules present on the surface of glioma
cells play a number of major roles in glioma cell migration.13,14,45
Fucose-containing glycans with potential clinical applications
are hypothesized as combating the development of malignant
gliomas. Indeed, it has been known for a long time that, under
normal circumstances, the astrocyte number remains constant
in the mammalian central nervous system during adulthood and
old age as a result of the balance of division promoters and
division inhibitors.46 Moreover, Nieto-Sampedro47 has identified
mitogen inhibitors as being immunologically related to blood
group oligosaccharides (i.e. Lewis antigen-related structures)
and the glycan epitopes of the epidermal growth factor receptor.
On the basis of these data, Aguilera et al.13 have synthesized
fucosyl-LacNAc-related structures, a family of oligosaccharides
with a common Lewis-X-type structure. These compounds
display a significant level of antiproliferative activity against
malignant glioblastoma cells.14 Our recent study also revealed
that cimetidine (a H2 receptor antagonist6) slightly, but never-
theless significantly, decreases the levels of expression of
endogenous receptors for fucose moieties;7 when combined with
temozolomide this cimetidine-induced decrease in endogenous
ligands for fucose gives rise to therapeutic benefits comparable
to those brought about by temozolomide alone in the case of
the U373 human glioblastoma model.7 The present study shows
that, in vitro, a â-D-fucopyranosyl-steroid (compound 19)
significantly modifies the levels of migration of human glio-
blastoma cells, but not of human NSCLC and prostate cancer
cells. Whether compound 19 could endow temozolomide with
additional therapeutic effects in vivo in the case of human
glioblastoma xenografts remains to be seen.

In ViVo Therapeutic Benefits from a Lactosylated Steroid
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1805
Scheme 4
Scheme 3
(a) 1-(R)-Bromoperbenzoylglucose, AgOTf, allylsilane, 4 Å molecular
sieves, CH2Cl2/toluene; (b) NaOMe, MeOH.
Experimental Section
(a) 1-(R)-Bromoperbenzoylfucose, AgOTf, allylsilane, 4 Å molecular
Chemistry. The deacetylation of the commercially available 16-
dehydropregnenolone acetate 1 and its protection as tert-butyl-
diphenylsilylether gives compound 2 in two steps with a 90%
overall yield (Scheme 1). The treatment of 2 with a large excess
of the organomagnesium derivative of 1-bromo-2,5-dimethoxyben-
zene in the presence of copper iodide led to a 1,4-addition to enone
2 to give compound 3 (Scheme 1). The silyl group (TBDPS) was
selectively removed by treatment with tetrabutylammonium fluoride
in tetrahydrofuran to give compound 4 (Scheme 1). In the 1H NMR
spectrum (in CDCl3) of isolated compound 4 the signal appearing
at δ 2.88 (d, J17-16 ) 9.6 Hz) was assigned to H-17 with the
assistance of the 1H-1H COSY and HMBC spectra. The coupling
constant of the signal revealed the transconfiguration of the
substituents carried by carbons C-16 and C-17. The NOESY plot
showed interactions between the H-18 and the H-21 protons, so
indicating the â-orientation of the methyl ketone (carried by C-17).
The interaction between the H-16 and the H-18 protons confirmed
the R-orientation of the 2,5-dimethoxyphenyl substituent (carried
by C-16). After the reduction of the ketone function of 4 in the
presence of sodium borohydride in methanol, a mixture of diaste-
reoisomers 5 (â-ol 5a and R-ol 5b; Scheme 2) was obtained (20â/
sieves, CH2Cl2/toluene; (b) NaOMe, MeOH.
biosyl, isomaltosyl, lactosyl) that we obtained. After the deprotec-
tion51 of the acetates or benzoates of the glycosylated compounds
with sodium methoxide in methanol we obtained 10 monoglyco-
sylated (16-25; Scheme 3 and Table 1A) and four bis-glycosylated
(27-30; Scheme 4 and Table 1B) final products.
The experimental procedures are detailed in the Supporting
Information.
Pharmacology
Cell Lines and Culture Media. Six human cancer cell lines
were used in the present work, namely, the Hs683 and
U-373MG glioblastoma, the HCT-15 colon cancer, the PC-3
prostate cancer, the A549 non-small-cell-lung cancer (NSCLC),
and the MCF-7 breast cancer cell lines. The manner in which
we handled these cell lines in our laboratory is detailed in the
Supporting Information.
Cytotoxicity Assay. Cytotoxicity analyses were performed
by means of the colorimetric MTT assay, as previously
detailed.5,52,53 The experimental protocol is given in the Sup-
porting Information.
Cell Migration Assay. The effects of various of the
compounds synthesized during the course of the present research
were analyzed at cell migration level on three different cell lines
including the PC-3 prostate, the A549 NSCLC, and the U373
glioblastoma cancer cells (see above). The cancer cell migration
levels were characterized quantitatively on a computer-assisted
device, as detailed elsewhere.5,7,54 This device enables the
trajectories of culture-maintained living cells to be quantified.
The greatest linear distance migrated by each cell was calculated
from these trajectories. This distance is in fact the maximum
relative distance from the point of origin, i.e. the MRDO
quantitative variable.5,7,54 The experiments were all performed
over 24 h, and one image was recorded every 4 min. Since the
analyses were carried out in triplicate, a minimum of 121 and
a maximum of 184 cells were analyzed in each experimental
condition. The influence of the various glycosylated steroids
from the present study on human cancer cell migration was
analyzed at 1, 10, and 100 nM.
1
20R ratio of 1/4 determined by H NMR and HPLC analyses).
The glycosylation of the sterol derivatives was performed using
the Koenigs-Knorr procedure.48 The preferred conditions utilized
glycosyl halides49 as donors and the sterol under silver50 (method
A) or mercuric51 (method B) catalysis in dry dichloromethane. When
silver trifluoromethane sulfonate catalysis was used, allylsilane was
added to the reaction mixture in order to trap the released triflic
acid. Acceptable yields of protected glycosides were obtained with
the â-configuration only. Starting from sterol 4, this synthesis led
first of all to the â-D-glucoside compound 6 (Scheme 3). Using
this general procedure, we condensed several mono- or disaccha-
rides to the hydroxyl function of 4 to obtain 10 additional
glycosylated steroids 6-15 (see Supporting Information). Com-
pounds 6-15 were not submitted to antimigratory analyses because
their glycoside moieties are still protected. From diol 5 (a mixture
of diastereoisomers 5a and 5b) we obtained the bis-â-L-fucoside
derivative 26 (Scheme 4) along with three other protected bis-
disaccharide derivatives that were not isolated any further. All the
H-17, H-18, H-20, and H-21 signals in the 1H NMR (in CDCl3) of
isolated compound 26 were assigned with the assistance of the 1H-
1H COSY and HMBC spectra. The NOESY plot showed interac-
tions between the H-18 protons and the H-20 and H-21 ones, with
H-17 indicating the S stereochemistry at C-20. This NMR analysis
showed that only the bis-fucosylated isomer of the R-ol 5a
derivative was isolated with a good yield. The other bis-fucosylated
isomer of the â-ol 5b derivative could not be isolated, probably
because the amount was too small. This result was also confirmed
in the case of the three other bis-glycosylated compounds (cello-
In Vivo Testing. Maximum Tolerated Dose. The maximum
tolerated dose (MTD) of a drug is defined as the maximum
dose which can be administered acutely (i.e. in one ip, single
dose) to healthy animals (i.e. not grafted with tumors). The
survival periods and the weights of the animals are recorded

1806 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Ingrassia et al.
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for up to 28 days postinjection. The detailed experimental
protocols are given in the Supporting Information.
The P388 Subcutaneous Lymphoma Model. The P388
lymphoma model was developed in our laboratory, as detailed
elsewhere.19 Details of the treatments are provided in the
captions to the figures. Additional information is also provided
in the Supporting Information. The potential gain in survival
obtained via a potential antitumor compound was evaluated by
means of the T/C index. This index is the ratio between the
median survival time of the treated animal group (T; n ) 9)
and that of the control group (C; n ) 9).
The Orthotopic U373 Human Glioblastoma Xenograft. In
vivo orthotopic xenografts of human U373 glioblastoma cells
in nude mice were obtained as previously detailed.7 The detailed
protocol is given in the Supporting Information.
Statistical Analyses. Statistical comparisons between control
and the treated groups were made by first carrying out the
Kruskal-Wallis test (a nonparametric one-way analysis of
variance), and, where this test revealed significant differences,
we investigated whether any of the groups treated differed from
control. For this purpose we applied the Dunn multiple
comparison procedure (2-sided test) adapted to the special case
of the comparisons of treatments and control, i.e. where only
(k - 1) comparisons were carried out among the k groups tested
by the Kruskal-Wallis test (instead of the possible k(k - 1)/2
comparisons considered in the general procedure). The levels
of statistical significance associated with the T/C-related survival
indices were determined by using Gehan’s generalized Wilcoxon
test. All these statistical analyses were carried out using Statistica
(Statsoft, Tulsa, OK).
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H.; Kaltner, H.; Hadari, Y.; Ruchoux, M. M.; Brotchi, J.; Zick, Y.;
Salmon, I.; Gabius, H. J.; Kiss, R. Galectins are differentially
expressed in supratentorial pilocytic astrocytomas, astrocytomas,
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tumor astrocyte migration. Brain Pathol. 2001, 11, 12-26.
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H.; Musette, S.; Darro, F.; Danguy, A.; Salmon, I.; Gabius, H. J.;
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E. F.; St-Pierre, Y. Upregulation of galectin-7 in murine lymphoma
is associated with progression toward an aggressive phenotype.
Leukemia 2003, 17, 751-759.
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galectin-7: Promoting tumorigenesis by up-regulating MMP-9 gene
expression. Cancer Res. 2005, 65, 5205-5210.
(27) Saussez, S.; Kiss, R. Galectin-7. Cell. Mol. Life Sci., in press.
(28) Branle, F.; Lefranc, F.; Camby, I.; Jeuken, J.; Geurts-Moespot, A.;
Sprenger, S.; Sweep, F.; Kiss, R.; Salmon, I. Evaluation of the
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Acknowledgment. The present work has been financed by
grants awarded by the “Re´gion de Bruxelles-Capitale” (Brussels,
Belgium).
Supporting Information Available: The analyses covering all
the compounds used in this work (melting points, elemental
analyses, optical rotations, HPLC analyses, 1H and 13C NMR, mass
spectra, and IR spectral data) and the pharmacology of the P388
subcutaneous model and the orthotopic U373 human glioblastoma
model. This material is available free of charge via the Internet at
http://pubs.acs.org.
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