FGF RECEPTOR-ACTIVATING N-SULFATE OLIGOSACCHARIDES,
PREPARATION THEREOF, AND THERAPEUTIC USE THEREOF
The present invention relates to N-sulfate oligosaccharides that are
agonists of the FGF/FGFR system, and to their preparation and therapeutic use.
Angiogenesis is a process of generation of new blood capillaries. During
the blockage of a blood vessel, angiogenesis, combined with arteriogenesis
(dilation of the capillaries), improves the revascularization of the blocked area. It
has been shown in vitro and in vivo that several growth factors, such as
Fibroblast Growth Factors (FGFs), stimulate the neovascularization process.
FGFs are a family of 23 members. FGF2 (or basic FGF) is an 18 kDa
protein. FGF2 induces, in endothelial cells in culture, their proliferation and
migration and the production of proteases, in vivo, FGF2 promotes
neovascularization. FGF2 interacts with endothelial cells via two classes of
receptors, the high-affinity receptors with tyrosine kinase activity (FGFRs) and
the low-affinity receptors of heparan sulfate proteoglycan (HSPG) type.
It is known that cell surface receptors with tyrosine kinase activity
associate in dimeric form with a complex formed from two ligand molecules and
one heparan sulfate molecule. The formation of this complex triggers a cascade
of intracellular signals resulting in activation of cell proliferation and migration,
which are two key processes involved in angiogenesis.
Thus, FGF2 and its receptors represent very pertinent targets for
therapies directed towards activating or inhibiting angiogenesis processes.
Synthetic oligosaccharides have also been the subject of studies of
interactions with the FGF receptors and have shown their inhibitory effects on
the binding of FGF-2 to its receptor on smooth muscle cells, with an IC50 of
16µg/mL, and also an inhibition of proliferation of these cells induced with
FGF-2, with an IC50 of about 23 µg/mL (C. Tabeur et al., Bioorg. & Med. Chem.,
1999, 7, 2003-2012; C. Noti et al., Chem. Eur. J., 2006, 12, 8664-8686).
We have now found novel synthetic oligosaccharides that are capable of
facilitating the formation of the FGF/FGFR complex and of thus promoting the in
vitro survival of endothelial cells and of increasing the in vitro and in vivo
formation of new blood vessels.
One subject of the present invention is novel oligosaccharide compounds
corresponding to formula (I):

in which:
- R1 represents a group -OSO3" or a hydroxyl group,
- R2 represents either a group -O-alkyl, or a monosaccharide of formula
(II), in which R represents an alkyl group:

- R3 represents a disaccharide of formula (III):

in which:
- R4 represents a group -OSO3" or a hydroxyl group,
- R5 represents a disaccharide of formula (IV):

in which:
- R6 represents a group -OSO3" or a hydroxyl group,
- R7 represents either a hydroxyl group or a disaccharide of formula (VI):

in which:
- R8 represents a group -OSO3 or a hydroxyl group,
- R9 represents either a hydroxyl group or a group -O-alkyl, or a
disaccharide of formula (VII):

in which R10 represents a group -O-alkyl,
on condition that: Rg represents a hydroxyl group or a group -O-alkyl when
R2 represents a monosaccharide of formula (II) as defined above; R7 represents
a disaccharide of formula (VI) as defined above when R2 represents a group
-O-alkyl; and R1, R4, R6 and R8 do not simultaneously represent hydroxyl groups.
In the context of the present invention, and unless otherwise mentioned in
the text, the term "alkyl group" is understood to mean a linear or branched
saturated aliphatic group comprising 1 to 4 carbon atoms. Examples that may be
mentioned include methyl, ethyl, propyl, isopropyl, butyl, isobutyl and fe/f-butyl
groups. In the compounds according to the invention, including each of the
subgroups of compounds that will be defined hereinbelow, the alkyl groups
advantageously represent methyl groups, except for the substituents R9 and R10
in which the alkyl radicals of the groups -O-alkyl advantageously represent
propyl groups.
The compounds according to the invention are synthetic oligosaccharides,
i.e. they are compounds obtained by total synthesis starting from intermediate
synthons, as will be described in detail in the text hereinbelow. In this respect,
they differ from oligosaccharides obtained by depolymerization or isolation from
complex mixtures of polysaccharides, such as heparins or low molecular weight
heparins. In particular, the compounds according to the invention have a well-
defined structure resulting from their chemical synthesis and are in the form of
pure oligosaccharides, i.e. they are free of other oligosaccharide species.
The invention encompasses the compounds of formula (I) in acid form or
in the form of any pharmaceutically acceptable salt thereof. In the acid form, the
functions -COO" and -SO3- are, respectively, in -COOH and -SO3H form.
The term "pharmaceutically acceptable salt of the compounds of the
invention" means a compound in which one or more of the functions -COO"
and/or -SO3- are ionically linked to a pharmaceutically acceptable cation. The
preferred salts according to the invention are those in which the cation is chosen
from alkali metal cations, especially the Na+ cation.
The compounds of formula (I) according to the invention also comprise
those in which one or more hydrogen or carbon atoms have been replaced with
a radioactive isotope, for example tritium or carbon 14C. Such labelled
compounds are useful in research, metabolism or pharmacokinetic studies, as
ligands in biochemical tests.
In formula (I) of the compounds according to the present invention, it is
understood that:
- the monosaccharide of formula (II) is linked to the disaccharide unit
represented in formula (I) via the oxygen atom located in position 4 of its uronic
acid unit,
- the disaccharide of formula (III) is linked to the disaccharide unit
represented in formula (I) via the oxygen atom located in position 1 of its
glucosamine unit,
- similarly, the disaccharide of formula (IV) is linked to the disaccharide of
formula (ill) via the oxygen atom located in position 1 of its glucosamine unit,
- similarly, the disaccharide of formula (VI) is linked to the disaccharide of
formula (IV) via the oxygen atom located in position 1 of its glucosamine unit,
- similarly, the disaccharide of formula (VII) is linked to the disaccharide of
formula (VI) via the oxygen atom located in position 1 of its glucosamine unit.
The term "glucosamine unit" means the monosaccharide unit having the
following formula:

The other type of saccharide unit present in the compounds according to
the invention is a uronic acid, more specifically an iduronic acid, corresponding
to the following formula:

Thus, the compounds of formula (I) according to the invention may also
be represented according to formula (I') as follows, in which the iduronic units
and the glucosamine units succeed each other and in which R1, R2, R4, R6 and
R7 are as defined previously:
Depending on the meanings of R2 and R7, the oligosaccharides according
to the invention may thus comprise from 7 to 10 saccharide units.
Among the compounds of formula (l)/(l') that are subjects of the invention,
mention may be made of those in which:
- R1, R3, R4, R5 and R6 are as defined previously,
- R2 represents a monosaccharide of formula (II) as defined previously,
and
- R7 represents a hydroxyl group.
Such compounds are heptasaccharides. They correspond to formula (l-1)
below, in which R7 represents a hydroxyl group and R, R1, R4 and R6 are as
defined previously, and are in acid form or in the form of any pharmaceutically
acceptable salt thereof.
Among the compounds of formula (l)/(l') that are subjects of the invention,
mention may be made of a subgroup of compounds in which R2 represents a
group -O-alkyl.
Such compounds are octasaccharides or decasaccharides. They
correspond to formula (I-2) below in which R1, R4, R6, R8 and R9 are as defined
previously and R2 represents a group -O-alkyl, and are in acid form or in the form
of any pharmaceutically acceptable salt thereof.
Among the compounds of formula (l)/(l') that are subjects of the invention,
mention may be made of a subgroup of compounds in which:
- R2 represents a group -O-alkyl, and
- R7 represents a disaccharide of formula (VI) as defined above, in which
R9 represents a disaccharide of formula (VII) as defined above.
Such compounds are decasaccharides. They correspond to formula (l-3)
below, in which R1, R4, R6, R8 and R10 are as defined previously, and are in acid
form or in the form of any pharmaceutically acceptable salt thereof.
Among the compounds of formula (I) that are subjects of the invention,
mention may be made of a subgroup of compounds in which:
- R2 represents a group -O-alkyl, and
- R7 represents a disaccharide of formula (VI) as defined above, in which
R9 represents either a hydroxyl group or a group -O-alkyl.
Such compounds are octasaccharides and correspond to formula (l-2)
above in which R1, R4, R6 and R8 are as defined previously, R2 represents a
group -O-alkyl and R9 represents either a hydroxyl group or a group -O-alkyl.
Advantageously, the octasaccharides according to the invention are such
that R9 represents a group -O-alkyl.
Other subgroups of compounds according to the invention may have
several of the characteristics listed above for each of the subgroups defined
previously.
The invention relates especially to the following oligosaccharides:
- methyl (sodium 4-O-propyl-2-O-sodium sulfonato-a-L-idopyranosyl-
uronate)-(1?4)-(2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-
glucopyranosyl)-(1?4)-[(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-
(1?4)-(2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-gluco-
pyranosyl-(1?4)]2-(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-
(1?4)-2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-
glucopyranoside (No. 1);
- methyl (sodium 4-O-propyl-2-O-sodium sulfonato-a-L-idopyranosyl-
uronate)-(1?4)-(2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-
glucopyranosyl)-(1?4)-[(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-
(1?4)-(2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-gluco-
pyranosyl-(1?4)]3-(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-
(1?4)-2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-gluco-
pyranoside (No. 2);
- sodium [methyl (sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-
(1?4).(2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-
glucopyranosyl)-(1?4)-[(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-
(1?4)-(2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-
glucopyranosyl)-(1?4)]2-2-O-sodiumsulfonato-a-L-idopyranoside]-uronate
(No. 3);
- methyl (sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-
(2-deoxy-2-sodium (sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-
sodium suifonato-a-L-idopyranosyiuronate)-(1?4)-(2-deoxy-2-sodium (sulfonato-
amino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-sodium sulfonato-a-L-
idopyranosyluronate)-(1?4)-(2-deoxy-6-O-sodium sulfonato-2-sodium
(sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-sodium sulfonato-a-L-
idopyranosyluronate)-(1?4)-2-deoxy-6-O-sodium sulfonato-2-sodium
(sulfonatoamino)-a-D-glucopyranoside (No. 4);
- methyl (sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-
(2-deoxy-2-sodium (sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-
sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-deoxy-6-O-sodium
sulfonato-2-sodium (sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-
sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-deoxy-2-sodium
(sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-sodium sulfonato-a-L-
idopyranosyluronate)-(1?4)-2-deoxy-6-O-sodium sulfonato-2-sodium
(sulfonatoamino)-a-D-glucopyranoside (No. 5);
- methyl (sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-
(2-deoxy-6-O-sodiumsulfonato-2-sodium (sulfonatoamino)-a-D-glucopyranosyl)-
(1?4)-(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-deoxy-
2-sodium (sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-sodium
sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-deoxy-2-sodium(sulfonatoamino)-
ot-D-glucopyranosyl)-(1?4)-(sodium 2-O-sodium sulfonato-a-L-idopyranosyl-
uronate)-(1?4)-2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-
glucopyranoside (No. 6);
- methyl (sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-
(2-deoxy-6-O-sodiumsulfonato-2-sodium (sulfonatoamino)-a-D-glucopyranosyl)-
(1?4)-(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-deoxy-
2-sodium (sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-sodium
sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-deoxy-6-O-sodium sulfonato-2-
sodium (sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-sodium
sulfonato-a-L-idopyranosyluronate)-(1?4)-2-deoxy-6-O-sodium sulfonato-2-
sodium (sulfonatoamino)-a-D-glucopyranoside (No. 7);
- methyl (sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-
(2-deoxy-6-O-sodium sulfonato-2-sodium(sulfonatoamino)-a-D-glucopyranosyl)-
[(1?4)-(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-deoxy-
6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-glucopyranosyl)]2-(1?4)-
(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-2-deoxy-6-O-
sodium sulfonato-2-(sulfonato)amino-a-D-glucopyranoside (No. 8);
- methyl (sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-
(2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonato)amino-a-D-glucopyranosyl)-
[(1?4)-(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-deoxy-
6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-glucopyranosyl)]2-(1?4)-
(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-2-deoxy-2-
sodium (sulfonatoamino)-a-D-glucopyranoside (No. 9).
In its principle, the process for preparing the compounds according to the
invention uses di- or oligosaccharide syr.thons prepared as reported previously
in the literature. Reference will be made especially to the patents or patent
applications EP 0 300 099, EP 0 529 715, EP 0 621 282 and EP 0 649 854, and
also to the publication by C. Van Boeckel and M. Petitou published in Angew.
Chem. Int. Ed. Engl., 1993, 32, 1671-1690. These synthons are then coupled
together so as to give an entirely protected equivalent of a compound according
to the invention. This protected equivalent is then converted into a compound
according to the invention. In the coupling reactions mentioned above, a "donor"
di- or oligosaccharide, activated on its anomeric carbon, reacts with an
"acceptor" di- or oligosaccharide, bearing a free hydroxyl.
The present invention thus relates to a process for preparing compounds
of formula (l)/(l'), characterized in that:
- in a first phase, a fully protected equivalent of the desired compound (I)
is synthesized,
- in a second phase, the groups -COO" and -OSO3" are introduced and/or
unmasked,
- in a third phase, the whole compound is deprotected, and
- in a fourth phase, the N-sulfate groups are introduced.
The synthesis of the fully protected equivalent of the desired compound (I)
is performed according to reactions that are well known to those skilled in the
art, and using methods for the synthesis of oligosaccharides (for example G.J.
Boons, Tetrahedron (1996), 52, 1095-1121 and patent applications
WO 98/03554 and WO 99/36443), in which a glycoside bond-donating
oligosaccharide is coupled with a glycoside bond-accepting oligosaccharide to
give another oligosaccharide whose size is equal to the sum of the sizes of the
two reactive species. This sequence is repeated until the compound of formula
(l)/(l') is obtained, optionally in protected form. The nature and profile of the
charge of the final desired compound determine the nature of the chemical
species used in the various synthetic steps, according to the rules well known to
those skilled in the art. Reference may be made, for example, to C. Van Boeckel
and M. Petitou, Angew. Chem. Int. Ed. Engl. (1993), 32, 1671-1690 or
alternatively to H. Paulsen, "Advances in selective chemical syntheses of
complex oligosaccharides", Angew. Chem. Int. Ed. Engl. (1982), 21, 155-173.
The compounds of the invention may naturally be prepared using various
strategies known to those skilled in the art of oligosaccharide synthesis. The
process described above is the preferred process of the invention. However, the
compounds of formula (l)/(l') may be prepared via other well-known methods of
sugar chemistry, described, for example, in "Monosaccharides, their chemistry
and their roles in natural products", P.M. Collins and R.J. Ferrier, J. Wiley &
Sons (1995) and by G.J. Boons in Tetrahedron (1996), 52, 1095-1121.
The protecting groups used in the process for preparing the compounds
of formula (l)/(l') are those that make it possible firstly to protect a reactive
function such as a hydroxyl or an amine during a synthesis, and secondly to
regenerate the intact reactive function at the end of the synthesis. The protecting
groups commonly used in sugar chemistry, as described, for example, in
"Protective Groups in Organic Synthesis", Greene et al., 3rd edition (John Wiley
& Sons, Inc., New York) are used to perform the process according to the
invention. The protecting groups are chosen, for example, from acetyl,
halomethyl, benzoyl, levulinyl, benzyl, allyl, ferf-butyldiphenylsilyl (tBDPS)
groups.
Activating groups may also be used; these are the groups conventionally
used in sugar chemistry, for example according to G.J. Boons, Tetrahedron
(1996), 52, 1095-1121. These activating groups are chosen, for example, from
imidates and thioglycosides.
The process described above allows the compounds of the invention to
be obtained in the form of salts, advantageously in the form of the sodium salt.
To obtain the corresponding acids, the compounds of the invention in salt form
may be placed in contact with a cation-exchange resin in acidic form. The
compounds of the invention in acid form may then be neutralized with a base to
obtain the desired salt. For the preparation of the salts of the compounds of
formula (l)/(l'), any mineral or organic base that gives pharmaceutically
acceptable salts with the compounds of formula (l)/(l') may be used.
A subject of the invention is also the compounds of formula 20A below, in
which Pg, Pg' and Pg", which may be identical or different, represent protecting
groups:

Such compounds are useful as intermediates in the synthesis of the
compounds of formula (I)/(!').
In particular, a subject of the invention is the compounds 20A in which Pg,
Pg' and Pg" represent, respectively, benzyl, allyl and acetyl groups. Such a
compound corresponds to disaccharide 20 illustrated in scheme 2 below, useful
for the synthesis of compounds 1 and 2 according to the invention, as will be
detailed hereinbelow:

The examples that follow describe the preparation of certain compounds
and synthetic intermediates in accordance with the invention. These compounds
are not limiting, but serve merely to illustrate the present invention. The starting
compounds and the reagents, when their mode of preparation is not expressly
described, are commercially available or described in the literature, or else may
be prepared according to methods that are described therein or that are known
to those skilled in the art.
The following abbreviations are used:
[a]D: optical rotation
Ac: acetyl
All: allyl
Bn: benzyl
Bz: benzoyl
TLC: thin-layer chromatography
CrO3: chromium trioxide
DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
ESI: Electron-Spray Ionization
h: hours
H2SO4: sulfuric acid
Lev: levulinyl
Me: methyl
min: minutes
Rf: Retardation factor (retention time measured on TLC relative to the
solvent migration front)
tBDPS: tert-butyldiphenylsilyl
Z: benzyloxycarbonyl
Preparation of the synthetic intermediates:
(Benzyl 2-O-acetvl-3-O-benzvl-4-O-levulinovl-a-L-idopvranosyluronate)-
(1?4)-1,6-anhvdro-2-azido-3-O-benzvl-2-deoxv-B-D-glucopvranose(12)
To a solution of compound 11 (11.6 g, 16.2 mmol) (described in the
preparation of compound 8 of patent application WO 2006/021653) in anhydrous
dioxane (340 mL) are successively added 4-dimethylaminopyridine (2.12 g,
17.3 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6.5 g,
34.3 mmol) and levulinic acid (3.6 mL; 34.3 mmol). After stirring for 4 hours 30
minutes, the mixture is diluted with dichloromethane (1800 mL). The organic
phase is washed successively with aqueous 10% potassium hydrogen sulfate
solution, with saturated sodium chloride solution, with saturated aqueous sodium
hydrogen carbonate solution and then with water, dried over sodium sulfate,
filtered and then evaporated to dryness. The residue is purified by
chromatography on a column of silica gel (5/1 v/v toluene/acetone) to give 12.7 g
of compound 12.
TLC: Rf = 0.42, silica gel, 3/1 v/v toluene/acetone
(Benzyl 2-O-acetyl-3-O-benzyl-4-Q-levulinovl-a-L-idopyranosyluronate)-
(1?4)-1.6-di-O-acetvl-2-azido-3-O-benzvl-2-deoxv-g,B-D-alucopvranose (13)
To a solution of compound 12 (12.76 g, 16.2 mmol) in acetic anhydride
(160 mL) is added, at 0°C, trifluoroacetic acid (14.1 mL, 183 mmol). The reaction
medium is stirred for 16 hours at room temperature. After concentrating, the
mixture is co-evaporated with toluene. Purification of the residue by
chromatography on a column of silica gel (4/1 v/v toluene/acetone) gives 10.5 g
of compound 13.
TLC: Rf = 0.49, silica gel, (4/1 v/v toluene/acetone)
(Benzyl 2-O-acetvl-3-O-benzvl-4-O-levulinovl-g-L-idopyranosyluronate)-
(1?4)-6-O-acetvl-2-azido-3-O-benzvl-2-deoxv-a.B-D-qlucopvranose (14)
A solution of compound 13 (10.5 g, 12.0 mmol) and benzylamine (50 mL,
457 mmol) in diethyl ether (360 mL) is stirred at room temperature for 2 hours.
The reaction mixture is diluted with diethyl ether (2000 mL). The organic phase
is washed with cold aqueous 1 M hydrochloric acid solution and then with water,
dried over sodium sulfate, filtered and then concentrated to dryness. The residue
is chromatographed on a column of silica gel (3/1 v/v toluene/acetone) to give
7.14 g of compound 14.
TLC: Rf = 0.40, silica gel, 3/1 v/v toluene/acetone
(Benzyl 2-O-acetvl-3-O-benzvl-4-O-levulinovl-a-L-idopvranosyluronate)-
(1?4)-6-O-acetvl-2-azido-3-O-benzyl-2-deoxv-a,B-D-qlucopvranose
trichloroacetimidate (15)
Trichloroacetonitrile (4.3 ml_, 42.8 mmol) and caesium carbonate (1.89 g,
13.7 mmol) are added to a solution of compound 14 (7.14 g, 8.56 mmol) in
dichloromethane (160 ml_). After stirring for 30 minutes, the reaction medium is
filtered and then concentrated. The residue is purified by chromatography on a
column of silica gel (2/1 v/v toluene/acetone + 0.1% triethylamine) to give 7.0 g
of compound 15.
TLC: Rf = 0.37 and 0.28, silica gel, 2/1 v/v toluene/acetone
(Benzyl 2-O-acetvl-4-O-(allvloxv)carbonvl-3-Q-benzvl-a-L-
idopvranosyluronate)-(1-^4)-1,6-anhvdro-2-azido-3-O-benzyl-2-deoxy-B-D-
glucopyranose (16)
To a solution of compound 11 (11.7 g, 17.3 mmol) (described in the
preparation of compound 8 of patent application WO 2006/021653) in anhydrous
tetrahydrofuran are successively added, at 0°C, pyridine (14 mL; 173 mmol),
4-dimethylaminopyridine (2.12 g, 17.3 mmol) and allyl chloroformate (18.3 mL,
173 mmol). After stirring for 16 hours at room temperature, water (47 mL) is
added at 0°C. After stirring for 30 minutes, the mixture is diluted with ethyl
acetate (800 mL). The organic phase is washed successively with aqueous 10%
potassium hydrogen sulfate solution and water and then with saturated aqueous
sodium hydrogen carbonate solution and water, dried over sodium sulfate,
filtered and then evaporated to dryness. The residue is purified by
chromatography on a column of silica gel (2/1 v/v cyclohexane/ethyl acetate) to
give 11.4 g of compound 16.
TLC: Rf = 0.37, silica gel, 2/1 v/v cyclohexane/ethyl acetate.
(Benzyl 2-O-acetvl-4-O-allvl-3-Q-benzvl-a-L-idopvranosyluronate)-(1?4)-
1,6-anhvdro-2-azido-3-O-benzvl-2-deoxv-fi-D-glucopvranose (17)
Compound 16 (11.44 g, 15.1 mmol) is dissolved in tetrahydrofuran
(100 mL). Palladium acetate (67.6 mg, 0.30 mmol) and triphenylphosphine
(395 mg, 1.5 mmol) are added. After stirring for 2 hours at reflux, the reaction
medium is concentrated to dryness. The residue is purified by chromatography
on a column of silica gel (3/1 v/v cyclohexane/ethyl acetate) to give 8.0 g of
compound 17.
TLC: Rf = 0.33, silica gel, 2/1 v/v cyclohexane/ethyl acetate.
(Benzyl 2-O-acetvl-4-O-allvl-3-O-benzvl-a-i_-idopvranosyluronate)-(1?4)-
1 ,64U6-O-acetvl-2-azido-3-O-benzvl-2-deoxv-g-D-
qlucopyranosvn-(1?4)-(methvl 2-O-acetvl-3-O-benzvl-g-L-idopyranosyluronate)-
(1^4V6-O-acetvl-3-O-benzvl-2-([(benzvloxv)carbonvnaminoV2-deoxv-a-D-
qlucopyranoside (22)
A mixture of the imidate 15 (541 mg, 0.553 mmol), the glycosyl acceptor
21 (650 mg, 0.83 mmol) (prepared according to the method described in
Carbohydrate Research (1987), 167 67-75) and powdered 4 A molecular sieves
(412 mg) in a toluene/dichloromethane mixture (23 ml_, 20/3 v/v) is stirred under
an argon atmosphere for 1 hour at 25°C. The reaction mixture is cooled to -25°C
and a 1 M solution of fe/f-butyldimethylsilyl triflate in dichloromethane (82.5 uL)
is added to the reaction medium. After 15 minutes, the reaction medium is
neutralized by addition of solid sodium hydrogen carbonate. After filtering and
concentrating, the residue obtained is purified by size exclusion chromatography
(Sephadex® LH20, 120 x 3 cm, 1/1 v/v dichloromethane/ethanol) to give 746 mg
of compound 22.
TLC: Rf = 0.37, silica gel, 5/6 v/v cyclohexane/ethyl acetate.
Methyl (benzyl 2-O-acetvl-3-O-benzvl-g-L-idopvranosyluronate)-(1 -*4W6-
0-acetvl-2-azido-3-O-benzvl-2-deoxv-a-D-qlucopvranosyl)-(1?4)-(methvl 2-O-
acetvl-3-O-benzvl-a-L-idopvranosyluronate)-(1?4)-6-Q-acetvl-3-Q-benzvl-2-
(r(benzvloxv)carbonvnaminoV2-deoxv-a-D-qlucopvranosidef23)
To a solution of compound 22 (2.3 g, 1.44 mmol) in a 1/2 v/v
toluene/ethanol mixture (290 mL) is added hydrazine acetate (662.3 mg,
7.2 mmol). The reaction medium is stirred for 2 hours at room temperature. After
concentrating, the residue is purified by flash chromatography on a column of
silica gel (5/6 v/v cyclohexane/ethyl acetate) to give 1.84 g of compound 23.
TLC: Rf = 0.48, silica gel, 5/6 v/v cyclohexane/ethyl acetate.
Methvl (benzyl 2-O-acetvl-3-O-benzvl-4-O-levulinovl-g-L-
idopvranosyluronate)-(1-^4)-(6-O-acetvl-2-azido-3-O-benzvl-2-deoxv-a-D-
qlucopvranosvn-(1^4V(benzvl2-O-acetvl-3-O-benzvl-a-L-idopyranosyluronate)-
(1?4)-(6-O-acetvl-2-azido-3-O-benzvl-2-deoxv-a-D-qlucopyranosyl-(1?4)-
fmethvl2-O-acetvl-3-O-benzvl-a-L-idopyranosyluronateV(1?4)-6-O-acetvl-3-O-
benzvl-2-(rfbenzvloxv)carbonvllamino)-2-deoxv-a-D-qlucopvranoside(24)
A mixture of the glycosyl acceptor 23 (1.97 g, 1.12 mmol), the imidate 15
(1.63g, 1.66 mmol) and powdered 4 A molecular sieves (2.6 g) in a 1/1 v/v
dichloromethane/toluene mixture (75 ml_) is stirred under an argon atmosphere
for 1 hour at 25°C. The reaction mixture is cooled to -20°C and a 1 M solution of
terf-butyldimethylsilyl triflate in dichloromethane (250 uL) is added to the reaction
medium. After 10 minutes, the reaction medium is neutralized by addition of
solid sodium hydrogen carbonate. After filtering and concentrating, the residue
obtained is purified by size exclusion chromatography (Sephadex® LH20, 190 x
3.2 cm, 1/1 v/v dichloromethane/ethanol) to give 1.63 g of compound 24.
TLC: Rf = 0.33, silica gel, 3/1 v/v toluene/acetone.
Methvl (benzyl 2-O-acetvl-3-Q-benzvl-g-i_-idopvranosyluronateH1 -^4)-(6-
0-acetvl-2-azido-3-O-benzvl-2-deoxv-g-D-qlucopvranosyl)-(1?4)-(benzvl 2-O-
acetvl-3-O-benzvl-a-L-idopvranosyluronateV(1?4)-(6-Q-acetvl-2-azido-3-O-
benzvl-2-deoxv-a-D-qlucopvranosyl)-(1?4)-(methvl2-Q-acetvl-3-O-benzvl-g-L-
idopyranosyluronateH1->4)-6-O-acetvl-3-O-benzvl-2-
(r(benzvloxv)carbonvnamino)-2-deoxv-g-D-qlucopvranoside(25)
To a solution of compound 24 (1.7g, 0.73 mmol) in a 1/2 v/v
toluene/ethanol mixture (145 mL) is added hydrazine acetate (338 mg,
3.67 mmol). The reaction medium is stirred for 2 hours at room temperature.
After concentrating, the residue is purified by flash chromatography on a column
of silica gel (1/1 v/v cyclohexane/ethyl acetate) to give compound 25 (1.41 g).
TLC: Rf = 0.47, silica gel, 1/1 v/v cyclohexane/ethyl acetate.
Methyl (benzyl 2-O-acetvl-4-O-allvl-3-O-benzvl-a-L-idopvranosyluronate)-
(1?4)-f6-O-acetvl-2-azido-3-O-benzvl-2-deoxv-a-D-qlucopvranosyl)-(1-^4)-
r(benzvl2-O-acetvl-3-O-benzvl-a-L-idopyranosyluronate)-(1^4')-(6-O-acetvl-2-
azido-3-O-benzvl-2-deoxv-a-D-glucopvranosyl-(1-^4)1?-(methyl 2-O-acetvl-3-O-
benzvl-a-L-idopvranosyluronate)-(1-»4V6-O-acetvl-3-O-benzvl-2-
(r(benzvloxv)carbonvnamino)-2-deoxv-a-D-qlucopyranoside (26)
A mixture of the imidate 20 (0.681 mg, 0.74 mmol), the glycosyl acceptor
25 (1.10 g, 0.5 mmol), and powdered 4 A molecular sieves (0.555 g) in a 1/1 v/v
dichloromethane/toluene mixture (26 ml_) is stirred under an argon atmosphere
for 1 hour at 25°C. The reaction mixture is cooled to -20°C and a 1 M solution of
terf-butyldimethylsilyl triflate in dichloromethane (111 uL) is added. After
20 minutes, the reaction medium is neutralized by addition of solid sodium
hydrogen carbonate. After filtering through Celite® and concentrating, the
residue obtained is chromatographed on a size exclusion column (Sephadex®
LH20, 190 x 3.2 cm, 1/1 v/v dichloromethane/ethanol) to give successively
468 mg of octasaccharide 26 and 842 mg of a mixture containing the
hexasaccharide 25 and the octasaccharide 26.
This mixture (842 mg) is treated under the above conditions to give
compound 26 (513.6 mg) after treatment and column chromatography
(Sephadex® LH20, 190 x 3.2 cm, 1/1 v/v dichloromethane/ethanol).
The two fractions (468 mg and 513.6 mg) are combined and purified by
preparative HPLC chromatography on a column of silica gel (2/1 v/v
toluene/ethyl acetate) to give compound 26 (1.03 g).
TLC: Rf = 0.44, silica gel, 2/1 v/v toluene/ethyl acetate.
Methyl (methyl 4-O-allyl-3-O-benzvl-a-L-idopvranosyluronateH1 -»4)-(2-
azido-3-O-benzyl-2-deoxv-a-D-glucopyranosyl)-(1?4)-f(methvl 3-O-benzyl-a-L-
idopvranosyluronateM1->4H2-azido-3-Q-benzvl-2-deoxv-a-D-glucopyranosyl-
(1 ->4Yl£-(methyl 3-O-benzvl-a-L-idopvranosyluronateM1 ->4)-3-Q-benzvl-2-
{[(benzvloxv)carbonvnamino)-2-deoxv-a-D-qlucopyranoside (27)
To a solution of compound 26 (819 mg, 0.27 mmol) in a 2/3 v/v
dichloromethane/methanol mixture (83 ml_) containing 3 A molecular sieves
(10.3 g) is added, at 0°C, under an argon atmosphere a 1 M solution of sodium
methoxide in methanol (1.65 ml_). After 16 hours at -18°C, the reaction medium
is neutralized with Dowex® 50WX4 H+ resin. After filtering and concentrating, the
residue is purified by size exclusion chromatography (Sephadex® LH20, 120 x
3 cm, 1/1 v/v dichloromethane/ethanol) followed by chromatography on a column
of silica gel (4/3 v/v toluene/acetone) to give 605 mg of compound 27.
TLC: Rf = 0.41, silica gel, 4/3 v/v toluene/acetone.
Methyl (methyl 4-Q-allvl-3-O-benzvl-2-O-triethvlammonium sulfonato-a-L-
idopvranosyluronate)-(1?4)-(2-azido-3-Q-benzvl-2-deoxv-6-O-triethvlammonium
sulfonato-a-D-qlucopvranosvn-(1-^4)-r(methvl3-Q-benzvl-2-O-triethvlammonium
sulfonato-a-L-idopvranosyluronate)-(1?4)-(2-azido-3-Q-benzvl-2-deoxv-6-O-
triethvlammonium sulfonato-g-D-qlucopvranosyl-(1?4)1?-(methvl 3-O-benzyl-2-Q-
triethvlammoniumsulfonato-a-L-idopvranosyluronate)-(1?4)-3-Q-benzvl-2-
([(benzvloxy)carbonvnamino)-2-deoxv-6-O-triethvlammonium sulfonato-a-D-
qlucopyranoside (28)
Compound 27 (300 mg, 0.12 mmol) is dried by co-distillation of N,N-
dimethylformamide (3x10 ml_) and is then dissolved in A/,A/-dimethylformamide
(11 ml_). To this solution is added the sulfur trioxide-triethylamine complex
(902 mg; 4.98 mmol). The mixture is stirred for 16 hours at 55°C protected from
light and then neutralized with methanol (202 uL, 4.98 mmol). The reaction
medium is deposited on a column of Sephadex® LH20 gel (95 x 2 cm) eluted
with a 1/1 v/v dichloromethane/ethanol mixture to give compound 28 (426 mg).
TLC: Rf = 0.32, silica gel, 11/7/1.6/4 v/v/v/v ethyl acetate/pyridine/acetic
acid/water.
Methyl (lithium 4-Q-allvl-3-O-benzvl-2-Q-lithium sulfonato-a-L-
idopvranosyluronate)-n^4H2-azido-3-O-benzvl-2-deoxv-6-Q-lithium sulfonato-
g-D-qlucopvranosylH1 -^4H(lithium 3-Q-benzvl-2-O-lithium sulfonato-a-L-
idopvranosyluronateH1->4M2-azido-3-O-benzvl-2-deoxv-6-Q-lithium sulfonato-
g-D-qlucopyranosyl-d->4yi?-(lithium 3-O-benzvl-2-O-lithium sulfonato-a-L-
idopvranosyluronate)-(1?4)-3-Q-benzvl-2-(r(benzvloxv)carbonvnamino>-2-
deoxv-6-O-lithium sulfonato-g-p-qlucopyranoside (29)
To a solution of compound 28 (459 mg, 0.12 mmol) in a 1/1 v/v
tetrahydrofuran/methanol mixture (19 ml_) is added, at 0°C, a 0.7 M solution of
lithium hydroxide in water (7.6 ml_; qs final concentration of 0.2 M). After 1 hour
at 0°C and then 16 hours at room temperature, the reaction medium is cooled to
0°C, neutralized with acetic acid (305 uL) and then deposited on a Sephadex®
LH20 column (95 x 2 cm) eluted with a 4/1 v/v methanol/water mixture to give
compound 29 (368 mg).
TLC: Rf = 0.25, silica gel, 27/19/4.2/11 v/v/v/v ethyl acetate/pyridine/acetic
acid/water.
Methyl (sodium 4-O-npropvl-2-Q-sodium sulfonato-a-L-
idopvranosyluronate)-(1^4M2-amino-2-deoxv-6-O-sodium sulfonato-a-D-
qlucopyranosylMI->4)-rsodium 2-O-sodium sulfonato-g-L-idopyranosyluronate)-
(1?4)-(2-amino-2-deoxv-6-O-sodium sulfonato-a-D-glucopyranosyl-d ->4)1?-
(sodium 2-O-sulfonato-a-L-idopvranosyluronate)-(1?4)-2-amino-2-deoxv-6-Q-
sodium sulfonato-a-D-qlucopyranoside (30)
A solution of compound 29 (170 mg) in a 2/3 v/v te/f-butanol/water
mixture (27 mL) is treated under pressure of hydrogen (1 bar) in the presence of
10% palladium-on-charcoal (340 mg) at 30°C for 24 hours. After filtration
(Millipore® LSWP 5 urn filter), the reaction mixture is deposited on a column of
Sephadex® G25-fine (90 x 3 cm) eluted with aqueous 0.2 M sodium chloride
solution. The fractions containing the product are concentrated and desalified
using the same column eluted with water. After concentrating to dryness,
compound 30 (205 mg) is obtained.
Mass: "ESI" method, negative mode: theoretical mass = 2327.55;
experimental mass: 2239 a.m.u. (iduronic acids observed in COOH form).
Methyl (benzyl 2-O-acetvl-3-O-benzvl-4-O-levulinovl-a-L-
idopyranosyluronate)-(1?4)-(6-O-acetvl-2-aziclo-3-O-benzvl-2-cleoxv-a-D-
glucopvranosylH1->4H(benzyl 2-O-acetvl-3-O-benzvl-a-L-idopvranosyluronate)-
(1-^4)-(6-O-acetvl-2-azido-3-O-benzvl-2-deoxv-a-D-glucopyranosyl-(1-^4)l7-
(methyl 2-O-acetvl-3-O-benzvl-a-L-idopvranosyluronate)-(1?4)-6-O-acetyl-3-O-
benzvl-2-(r(benzvloxv)carbonvnamino>-2-deoxv-a-D-glucopyranoside (31)
A mixture of the imidate 15 (250 mg, 0.255 mmol), the glycosyl acceptor
25 (376 mg, 0.169 mmol) and powdered 4 A molecular sieves (396 mg) in a 1/1
v/v dichloromethane/toluene mixture (11.4 mL) is stirred under an argon
atmosphere for 1 hour at 25°C. The reaction mixture is cooled to -20°C and a
0.1 M solution of fe/f-butyldimethylsilyl triflate in dichloromethane (381 uL) is
added. After 12 minutes, the reaction medium is neutralized by addition of solid
sodium hydrogen carbonate. After filtering and concentrating, the residue
obtained is chromatographed on a Sephadex® LH20 column (120 x 3 cm, 1/1 v/v
dichloromethane/ethanol) to give successively 150 mg of crude compound 31
and 318 mg of a mixture containing the hexasaccharide 25 and the
octasaccharide 31.
The mixture containing the hexasaccharide 25 and the octasaccharide 33
is treated under the above conditions to give after size exclusion
chromatography (Sephadex® LH20) 151 mg of crude compound 31 and 191 mg
of a mixture containing the hexasaccharide 25 and the octasaccharide 31. This
operation is repeated twice to the point of depletion of the hexasaccharide 25.
The fractions containing the crude octasaccharide 31 are combined and
purified by flash chromatography on a column of silica gel (7/3 v/v toluene/ethyl
acetate) to give compound 31 (351 mg).
TLC: Rf = 0.37, silica gel, 7/5 v/v toluene/ethyl acetate.
Methyl (benzyl 2-O-acetvl-3-O-benzvl-a-L-idopyranosyluronateH1 ->4H6-
0-acetvl-2-azido-3-O-benzvl-2-deoxv-a-D-glucopyranosyl)-(1-^4)-r(benzvl 2-O-
acetvl-3-O-benzvl-a-L-idopvranosyluronate)-(1?4)-(6-O-acetvl-2-azido-3-Q-
benzvl-2-deoxv-a-D-qlucopvranosyl-(1-^4)1?-(methvl2-O-acetvl-3-O-benzvl-a-L-
idopyranosyluronateVd ->4)- 6-Q-acetvl-3-O-benzvl-2-
(r(benzvloxv)carbonvnamino)-2-deoxv-a-D-glucopyranoside (32)
To a solution of compound 31 (335 mg, 0.11 mmol) in a 1/2 v/v
toluene/ethanol mixture (22 ml_) is added hydrazine acetate (51 mg, 0.55 mmol).
The reaction medium is stirred for 2 hours at room temperature. The reaction
mixture is diluted with dichloromethane (50 ml_). The organic phase is washed
with aqueous 2% potassium hydrogen sulfate solution, and then with water,
dried over sodium sulfate, filtered and then concentrated to dryness. The residue
is purified by chromatography on a column of silica gel (7/3 v/v toluene/acetone)
to give compound 32 (238 mg).
TLC: Rf = 0.50, silica gel, 5/6 v/v cyclohexane/ethyl acetate.
Methyl (benzyl 2-O-acetvl-4-O-allvl-3-O-benzvl-a-L-idopvranosyluronate)-
(1?4)-(6-O-acetvl-2-azido-3-O-benzvl-2-deoxv-a-D-qlucopyranosyl)-f1->4)-
r(benzvl2-O-acetvl-3-O-benzvl-a-L-idopyranosyluronate)-(1?4)-(6-O-acetvl-2-
azido-3-O-benzvl-2-deoxv-a-D-qlucopvranosyl-(1-^4)V(methyl 2-O-acetvl-3-O-
benzvl-a-L-idopvranosyluronateH1->4)-6-O-acetvl-3-O-benzvl-2-
([(benzvloxv)carbonvllamino>-2-deoxv-a-D-glucopyranoside (33)
A mixture of the imidate 20 (64 mg, 0.066 mmol), the glycosyl acceptor 32
(130 mg, 0.044 mmol) and powdered 4 A molecular sieves (49 mg) in a 1/1 v/v
dichloromethane/toluene mixture (2.2 ml_) is stirred under an argon atmosphere
for 1 hour at 25°C. The reaction mixture is cooled to -20°C and a 0.1 M solution
of terf-butyldimethylsilyl triflate in dichloromethane (100 uL) is added. After
10 minutes, the reaction medium is neutralized by addition of solid sodium
hydrogen carbonate. After filtering and concentrating, the residue obtained is
chromatographed on a Sephadex® LH20 column (120 x 3 cm, 1/1 v/v
dichloromethane/ethanol) to give successively 61 mg of crude compound 33 and
87.7 mg of a mixture containing the octasaccharide 32 and the decasaccharide
33.
The above mixture (87.7 mg) is treated under the above conditions to give
crude compound 33 (120 mg) after chromatography on a Sephadex® LH20
column (120 x 3 cm, 1/1 v/v dichloromethane/ethanol).
The two fractions containing the crude compound are combined and
purified by flash chromatography on a column of silica gel (7/2 v/v toluene/ethyl
acetate) to give compound 33 (120 mg).
TLC: Rf = 0.52, silica gel, 1/1 v/v cyclohexane/ethyl acetate
Methyl (methyl 4-O-allvl-3-O-benzvl-a-L-idopvranosyluronateH1 -»4)-(2-
azido-3-O-benzvl-2-deoxv-g-D-glucopyranosylM1->4)-r(methvl 3-O-benzyl-g-L-
idopvranosyluronate)-(1?4)-(2-azido-3-O-benzyl-2-deoxv-a-D-glucopyranosyl-
(1 ->4Yl3-(methvl 3-O-benzvl-a-i_-idopvranosyluronateH1 ->4)-3-Q-benzvl-2-
{r(benzyloxv)carbonvnamino)-2-deoxv-a-D-glucopyranoside (34)
To a solution, cooled to 0°C, of compound 33 (51.8 mg, 0.014 mmol) in a
2/3 v/v dichloromethane/methanol mixture (982 uL) containing 3 A molecular
sieves (125 mg) is added under an argon atmosphere a 1 M solution of sodium
methoxide in methanol (21.0 uL). After 5 hours at room temperature, the
reaction medium is neutralized with Dowex® 50WX4 H+ resin. After filtering and
concentrating, the residue is purified by size exclusion chromatography
(Sephadex® LH20, 95 x 2 cm, 1/1 v/v dichloromethane/ethanol) to give
compound 34 (31.4 mg).
TLC: Rf = 0.47, silica gel, 7/3 v/v dichloromethane/acetone
Methyl (methyl 4-O-a[lyl-3-Q-benzvl-2-O-triethylammonium sulfonato-g-L-
idopyranosyluronate)-(1?4)-(2-azido-3-O-benzvl-2-cleoxy-6-O-triethvlammonium
sulfonato-a-D-qlucopyranosvO-(1?4)-r(methvl 3-O-benzyl-2-O-triethylammonium
sulfonato-a-L-idopvranosyluronate)-(1?4)-(2-azido-3-O-benzyl-2-deoxv-6-Q-
triethylammonium sulfonato-g-D-qlucopyranosyl-d -^4)V(methyl 3-O-benzyl-2-Q-
triethylammonium sulfonato-a-L-idopyranosyluronate)-(1?4)-3-O-benzvl-2-
(r(benzyloxy)carbonvnamino}-2-deoxv-6-O-triethylammonium sulfonato-g-D-
qlucopyranoside (35)
Compound 34 (31.5 mg, 10.6 umol) is treated as in the preparation of
compound 28 to give compound 35 (47.0 mg) after size exclusion
chromatography (Sephadex® LH20, 100 x 1.2 cm, 1/1 v/v
dichioromethane/methanol).
TLC: Rf = 0.55, silica gel, 16/11/2.6/7 v/v/v/v ethyl acetate/pyridine/acetic
acid/water.
Methyl (lithium 4-Q-allvl-3-O-benzyl-2-Q-lithium sulfonato-a-L-
idopyranosyluronate)-(1-^4)-(2-azido-3-O-benzyl-2-deoxv-6-O-lithium sulfonato-
g-D-glucopyranosyl)-(1 ->4H(lithium 3-O-benzyl-2-Q-lithium sulfonato-a-L-
idopvranosyluronate)-(1?4)-(2-azido-3-Q-benzvl-2-deoxv-6-O-lithium sulfonato-
g-D-qlucopyranosyl-d -»4%(lithium 3-Q-benzvl-2-O-lithium sulfonato-a-L-
idopvranosyluronate)-(1?4)-3-O-benzvl-2-(r(benzvloxv)carbonvllamino}-2-
deoxv-6-O-lithium sulfonato-g-D-glucopyranoside (36)
To a solution of compound 35 (47 mg, 12.5 umol) in a 1/1 v/v
tetrahydrofuran/methanol mixture (2.0 ml_) is added, at 0°C, a 1 M solution of
lithium hydroxide in water (500 uL; qs a final concentration of 0.2 M). After
1 hour at 0°C and then 4 hours at room temperature, the reaction medium is
neutralized with acetic acid and then left at -20°C for 16 hours. The reaction
mixture is deposited on a column of Sephadex® LH20 gel eluted with a 4/1 v/v
methanol/water mixture to give compound 36 (38.9 mg).
Methyl (sodium 4-O-propvl-2-Q-sodium sulfonato-a-L-
idopvranosyluronate)-(1?4)-(2-amino-2-deoxv-6-Q-sodium sulfonato-a-D-
qlucopyranosylMI ->4Msodium 2-O-sodium sulfonato-q-L-idopyranosyluronate)-
(1?4)-(2-amino-2-deoxv-6-Q-sodium sulfonato-a-D-glucopyranosyl-(1?4)1t-
(sodium 2-O-sodium sulfonato-a-L-idopvranosyluronate)-(1?4)-2-amino-2-
deoxy-6-O-sodium sulfonato-a-D-glucopyranoside (37)
A solution of compound 36 (40 mg, 10.8 umol) in a 6/9 v/v tert-
butanol/water mixture (1 mL) is treated under pressure of hydrogen (11 bar) in
the presence of 10% palladium-on-charcoal (80 mg, 2 x amount of compound to
be reduced) at 40°C for 4 hours. After filtration (Millipore® LSWP 5 urn filter), the
solution is deposited on a column of Chelex® 100 resin (1 mL) eluted with water
and then concentrated to dryness. The crude compound thus obtained (27 mg)
is used v\/ithout further purification in the following step.
Mass: "ESI" method, negative mode: theoretical mass = 2781.10;
experimental mass: 2782.05 ± 0.30 a.m.u.
Methyl rmethyl (methyl 2-O-acetvl-3-O-benzvl-4-O-levulinovl-a-L-
idopvranosyluronate)-(1?4)-(6-O-acetvl-2-azido-3-O-benzvl-2-deoxv-a-D-
glucopvranosvn-(1?4)-2-O-acetvl-3-O-benzvl-a-L-idopyranoside1uronate (40)
A mixture of the imidate 38 (1.1 g, 1.22 mmol), prepared according to the
method described by C. Tabeur et al., BioOrg. Med. Chem. (1999) 7, 2003-2012,
the glycosyl acceptor 39 (864 mg, 2.44 mmol), prepared according to the
method described by Koshida, S. et al., Tetrahedron Lett., 1999, 40, 5725-5728,
and powdered 4 A molecular sieves (914 mg) in toluene (43 mL) is stirred under
an argon atmosphere for 1 hour at 25°C. The reaction mixture is cooled to -20°C
and a 1 M solution of te/f-butyldimethylsilyl triflate in dichloromethane (183 uL) is
added. After stirring for 1 hour, the reaction medium is neutralized by addition of
solid sodium hydrogen carbonate. After filtering through Celite® and
concentrating, the residue obtained is chromatographed on a Sephadex® LH20
column (120 x 3 cm, 1/1 v/v dichloromethane/ethanol) followed by
chromatography on a column of silica gel (1/1 v/v toluene/ethyl acetate) to give
compound 40 (936 mg).
TLC: Rf = 0.38, silica gel, 1/1 v/v toluene/ethyl acetate
Methyl Tmethyl (methyl 2-O-acetvl-3-O-benzvl-a-L-idopyranosyluronate)-
(1?4)-(6-O-acetvl-2-azido-3-O-benzvl-2-deoxy-a-D-qlucopyranosyl)-(1?4)-2-O-
acetvl-3-O-benzvl-a-L-idopyranosideluronate (41)
Compound 40 (2.56 g, 2.34 mmol) is treated according to the same
procedure as that described for the preparation of compound 23 to give
compound 41 (2.17 g) after flash chromatography on a column of silica gel (1/1
v/v toluene/ethyl acetate).
TLC: Rf = 0.35, silica gel, 1/1 v/v toluene/ethyl acetate
Methyl fmethyl (methyl 2-Q-acetvl-3-Q-benzvl-4-Q-levulinovl-a-L-
idopvranosyluronateM1->4)-(6-O-acetvl-2-azido-3-O-benzvl-2-deoxv-a-D-
glucopvranosyl)-(1->4Mmethvl 2-O-acetvl-3-O-benzvl-a-L-idopyranosyluronate)-
(1?4)-(6-O-acetvl-2-azido-3-O-benzvl-2-deoxv-a-D-qlucopyranosyl)-(1?4)-2-O-
acetvl-3-Q-benzvl-a-L-idopvranosideluronate (42)
A mixture of the imidate 38 (1.1 g, 1.22 mmol), the glycosyl acceptor 41
(1.2 g, 1.20 mmol) and powdered 4 A molecular sieves (903 mg) in
dichloromethane (42 mL) is stirred under an argon atmosphere for 1 hour at
25°C. The reaction mixture is cooled to -20°C and a 1 M solution of tert-
butyldimethylsilyl triflate in dichloromethane (181 uL) is added. After 15 minutes,
the reaction medium is neutralized by addition of solid sodium hydrogen
carbonate. After filtering and concentrating, the residue obtained is purified by
chromatography on a size exclusion column (Sephadex® LH20, 190 x 3.2 cm,
1/1 v/v dichloromethane/ethanol) followed by chromatography on a column of
silica gel (1/1 v/v toluene/ethyl acetate) to give 1.63 g of compound 42.
TLC: Rf = 0.30, silica gel, 1/1 v/v toluene/ethyl acetate
Methyl Tmethvl (methyl 2-O-acetvl-3-O-benzvl-a-L-idopvranosyluronateV
(1?4)-(6-O-acetvl-2-azido-3-O-benzvl-2-deoxv-g-D-qlucopvranosvn-(1?4)-
(methyl 2-O-acetvl-3-O-benzvl-a-L-idopvranosyluronate)-(1?4)-(6-Q-acetvl-2-
azido-3-O-benzvl-2-deoxv-a-D-glucopvranosyl)-(1?4)-2-O-acetvl-3-O-benzyl-a-L-
idopyranosideluronate (43)
Compound 42 (3.90 g, 2.25 mmol) is treated according to the same
procedure as that described for the preparation of compound 23.
Chromatography on a column of silica gel (1/1 v/v toluene/ethyl acetate) gives
compound 43 (3.39 g).
TLC: Rf = 0.33, silica gel, 1/1 v/v toluene/ethyl acetate
(3.4-di-O-benzvl-2-O-(4-methoxv)benzvl-6-O-fe/t-butvldimethvlsilvl-a-L-
idopvranosyl)-(1?4)-1,6-anhvdro-2-azido-3-O-benzvl-2-deoxv-B-D-
qlucopyranose (45)
To a solution of compound 44 (32.3 g, 42.2 mmol) (described in the
preparation of compound 108 of patent application WO 2006/021653) in N,N-
dimethylformamide (210 ml_) is added, at 0°C and under argon, benzyl bromide
(25 mL, 211 mmol) and then 55% NaH (3 g, 126 mmol). After 20 minutes of
magnetic stirring, methanol is added (30 mL), the reaction medium is
concentrated under vacuum, and the crude reaction product is diluted with ethyl
acetate, washed with water and then with saturated aqueous sodium chloride
solution, dried (Na2S04), filtered and concentrated. The residue obtained is used
in the following step without purification.
LC-MS m/z 871.7 [(M + NH4)+]. TR = 13.86 min
(2-O-acetvl-3.4-di-O-benzvl-6-O-fe/f-butvldimethvlsilvl-a-L-idopvranosyl)-
(1?4)-1.6-anhvdro-2-azido-3-Q-benzvl-2-deoxv-B-D-qlucopvranose (46)
To a solution of crude compound 45 (38.6 g) in dichloromethane (1.6 L)
are added water (80 mL) and then, at 0°C, DDQ (14.2 g). After stirring for
4 hours 45 minutes at 0°C, the medium is diluted with dichloromethane and
aqueous sodium hydrogen carbonate solution is added. The organic phase is
then washed with water, dried (Na2S04), filtered and concentrated. The
compound obtained is used in the following step without purification.
The residue obtained is dissolved in dichloromethane (350 mL), and
triethylamine (13 mL), 4-dimethylaminopyridine (2 g) and acetic anhydride
(60 mL) are added. After magnetic stirring for 10 minutes at 0°C, and then 1
hour 45 minutes at room temperature, the reaction mixture is diluted with
dichloromethane, and then washed successively with aqueous 10% potassium
hydrogen sulfate solution, water, and the organic phase is then dried (Na2S04),
filtered and concentrated.
The residue obtained is purified on silica (ethyl acetate/cyclohexane) to
give compound 46 (26.8 g).
LC-MS m/z 798.3 [(M + Na)+]. TR = 12.97 min
(Methyl 2-O-acetvl-3,4-di-O-benzvl-a-L-idopvranosyluronate)-(1?4)-1,6-
anhvdro-2-azido-3-O-benzvl-2-deoxv-B-D-qlucopyranose (47)
To a solution of compound 46 (26.3 g, 33.9 mmol) in acetone (1.4 L) is
added, at 0°C, a solution of Cr03 (10.5 g) in aqueous 3.5 M H2S04 (47 rnL).
After mechanical stirring for 4 hours at 0°C, the reaction medium is diluted with
dichloromethane, washed with water until neutral, and the organic phase is then
dried (Na2S04), filtered and concentrated. The compound obtained is used in the
following step without purification.
The residue obtained is dissolved in A/,A/-dimethylformamide (210 mL),
and potassium hydrogen carbonate (17 g) and methyl iodide (21 mL) are added.
The reaction mixture is stirred at room temperature for 16 hours, and then
concentrated under vacuum. The residue is diluted with ethyl acetate and then
washed with water, with saturated aqueous sodium thiosulfate solution, with
saturated aqueous sodium chloride solution, and then dried (Na2S04), filtered
and concentrated. The compound obtained is used in the following step without
purification.
LC-MS m/z 707.3 [(M + NH4)+]. TR = 10.37 min
(Methyl 2-O-acetvl-3,4-di-Q-benzvl-a-L-idopvranosyluronate)-(1?4)-1.6-
di-O-acetvl-2-azido-3-O-benzvl-2-deoxv-a.B-D-glucopvranose (48)
The crude residue obtained in the preceding step is dissolved in acetic
anhydride (177 mL) and trifluoroacetic acid (TFA) (17.7 mL) is then added. The
reaction mixture is stirred for 16 hours, and is then concentrated, co-evaporated
with toluene and purified on silica gel (cyclohexane/ethyl acetate), to give
compound 48 (17.4 g).
LC-MS m/z 809.3 [(M + NH4)+]. TR = 10.81 min
(Methyl 2-O-acetvl-3,4-di-O-benzyl-a-L-idopvranosyluronate)-(1?4)-6-Q-
acetvl-2-azido-3-O-benzvl-2-deoxv-a.B-D-glucopyranose (49)
To a solution of compound 48 (7 g, 8.84 mmol) in diethyl ether (303 mL)
is added, at 0°C and under argon, benzylamine (BnNH2) (29.7 mL). After
magnetic stirring for 1 hour at 0°C and then for 6 hours at room temperature, the
reaction mixture is neutralized with cold aqueous 1N HCI solution, washed with
water, dried (Na2S04), filtered and concentrated, and purified on silica gel (ethyl
acetate/cyclohexane) to give compound 49 (5.95 g).
LC-MS m/z 767.7 [(M + NH4)+]. TR = 1.64 min
(Methyl 2-O-acefyl-3,4-di-O-benzvl-a-L-idopvranosyluronateH1->4)-6-O-
acetyl-^-azido-S-O-benzvl^-deoxv-a.B-D-glucopvranose trichloroacetimidate (50)
To a solution of compound 49 (5.94 g, 7.9 mmol) in dichloromethane
(150 mL) are added, under argon, caesium carbonate (CS2CO3) (4.1 g) and then
trichloroacetonitrile (CCI3CN) (3.9 mL). After stirring for 45 minutes at room
temperature, the reaction mixture is filtered and then concentrated. The residue
is purified on silica gel (ethyl acetate/cyclohexane + 0.1% triethylamine) to give
compound 50 (5.7 g).
LC-MS m/z 912.0 [(M + NhU)*]. TR = 1.81 min
Methyl (methyl (methyl 2-O-acetvl-3.4-di-O-benzvl-a-L-
idopvranosyluronate)-(1?4)-(6-O-acetvl-2-azido-3-O-benzyl-2-deoxv-a-D-
qlucopyranosylH1->4)-r(methvl 2-O-acetvl-3-O-benzvl-a-L-idopyranosyluronate)-
f1-^4)-(6-O-acetvl-2-azido-3-O-benzvl-2-deoxv-a-D-alucopvranosyl)-(1?4)1?-2-O-
acetvl-3-O-benzvl-a-L-idopyranoside>uronate (51)
A mixture of the glycosyl acceptor 43 (200 mg, 0.12 mmol), the imidate 50
(163 mg, 0.18 mmol) and powdered 4 A molecular sieves (137 mg) in
dichloromethane (6.5 mL) is stirred under an argon atmosphere for 1 hour at
25°C. The reaction mixture is cooled to -20°C and a 1 M solution of tert-
butyldimethylsilyl triflate in dichloromethane (27 uL) is added. After 1 hour, the
reaction medium is neutralized by addition of solid sodium hydrogen carbonate.
After filtering and concentrating, the residue obtained is purified by
chromatography on a size exclusion column (Sephadex® LH20, 90 x 3 cm, 1/1
v/v dichloromethane/ethanol) followed by chromatography on a column of silica
gel (2/1 v/v toluene/ethyl acetate) to give compound 51 (212 mg).
TLC: Rf = 0.38, silica gel, 1/1 v/v toluene/ethyl acetate.
Methyl (methyl (methyl 3,4-di-O-benzvl-a-i_-idopvranosyluronateM1->4)-
(2-azido-3-O-benzvl-2-deoxv-g-D-qlucopvranosyl)-(1?4)-r(methvl 3-O-benzvl-a-L-
idopyranosyluronate)-(1?4)-(2-azido-3-O-benzvl-2-deoxv-a-D-glucopyranosyl)-
(1?4)l?-3-O-benzvl-a-L-idopyranoside)uronate (52)
Compound 51 (271 mg, 0.11 mmol) is treated according to the same
procedure as that described for the preparation of compound 27 to give
compound 52 (186 mg) after chromatography on a size exclusion column
(Sephadex® LH20, 120 x 3 cm, 1/1 v/v dichloromethane/ethanol) followed by
chromatography on a column of silica gel (5/3 v/v toluene/acetone).
TLC: Rf = 0.65, silica gel, 4/3 v/v toluene/ethyl acetate
Methyl (methyl (methyl 3,4-di-O-benzvl-2-O-triethylammonium sulfonato-
a-L-idopvranosyluronate)-(1?4)-(2-azido-3-Q-benzvl-2-deoxv-6-O-
triethylammonium sulfonato-a-D-glucopvranosyl)-(1?4)-f(methyl 3-Q-benzvl-2-O-
triethvlammonium sulfonato-a-L-idopvranosyluronate)-(1->4H2-azido-3-O-
benzvl-2-deoxv-6-O-triethvlammonium sulfonato-a-D-qlucopvranosyl)-(1->4Yl?-3-
O-benzvl-2-O-triethvlammonium sulfonato-a-L-idopvranoside)uronate (53)
Compound 52 (172 mg, 0.08 mmol) is treated according to the same
procedure as that described for the preparation of compound 28 to give
compound 53 (196 mg) after size exclusion chromatography (Sephadex® LH20,
120 x 3 cm, 1/1 v/v dichloromethane/ethanol).
1H NMR (D20) 8 of the main anomeric protons: 5.41; 5.38; 5.34; 5.15;
5.14; 5.05;5.15; 5.02 ppm.
Lithium (methyl (lithium 3,4-di-Q-benzvl-2-O-lithium sulfonato-a-L-
idopvranosyluronate)-(1?4)-(2-azido-3-O-benzvl-2-deoxv-6-Q-lithium sulfonato-
g-D-qlucopvranosyl)-(1 ->4H(lithium 3-Q-benzvl-2-Q-lithium sulfonato-a-L-
idopvranosyluronate)-(1?4)-(2-azido-3-Q-benzvl-2-deoxv-6-O-lithium sulfonato-
a-D-qlucopvranosyl)-(1?4)1?-3-O-benzvl-2-O-lithium sulfonato-a-L-
idopyranosidejuronate (54)
Compound 53 (186 mg, 70.7 umol) is treated according to the same
procedure as that described for the preparation of compound 29 to give
compound 54 (141 mg) after size exclusion chromatography (Sephadex® LH20,
120 x 3 cm, 50/50/1 v/v/v dichloromethane/ethanol/water).
TLC: Rf = 0.24, silica gel, 27/19/4.2/11 v/v/v/v ethyl acetate/pyridine/acetic
acid/water
Sodium (methyl (sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-
(1?4)-(2-amino-2-deoxv-6-O-sodium sulfonato-a-D-glucopyranosvO-d ->4)-
lYsodium 2-O-sodium sulfonato-a-i_-idopvranosyluronate)-(1 -»4V(2-amino-2-
deoxy-6-O-sodium sulfonato-a-D-glucopyranosvD-(1?4)l?-2-Q-sodium sulfonato-
g-L-idopvranoside}uronate (55)
A solution of compound 54 (60.0 mg, 0.022 mmol) in a 1/1 v/v tert-
butanol/water mixture (6 ml_) is treated under pressure of hydrogen (.< 200 mbar)
in the presence of 10% palladium-on-charcoal (60 mg) at room temperature.
After stirring for 24 hours, the reaction medium is filtered (Millipore® LSWP 5 urn
filter) and then concentrated to dryness. The crude product 55 thus obtained
(48.7 mg) is used without further purification in the following step.
Methyl (methyl 2-O-acetvl-3-O-benzvl-4-Q-levulinovl-a-L-
idopvranosyluronate)-(1?4)-6-O-acetvl-3-O-benzvl-2-benzvloxvcarbonylamino-
2-deoxv-a-D-qlucopyranoside (57)
To a solution of compound 56 (23.5 g, 30 mmol; prepared according to
the method described in Carbohydrate Research (1987), 167, 67-75) in
dichloromethane (600 ml_) are added, at 0°C and under an inert atmosphere,
4-dimethylaminopyridine (733 mg, 6 mmol), 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (11.5 g, 60 mmol) and levulinic acid (6.2 ml_,
60 mmol). After stirring for 16 hours at room temperature, the mixture is diluted
with dichloromethane (1.5 L). The organic phase is washed successively with
aqueous 10% potassium hydrogen sulfate solution, with water, with saturated
sodium hydrogen carbonate solution and then with water, dried over sodium
sulfate, filtered and then evaporated to dryness. The residue is purified by flash
chromatography on a column of silica gel (1/3 cyclohexane/ethyl acetate) to give
compound 57 (22.6 g).
Rf = 0.37, silica gel, 1/3 v/v cyclohexane/ethyl acetate
Methyl (methyl 2-O-acetvl-3-O-benzvl-4-O-levulinovl-g-L-
idopvranosyluronate)-(1-^4)-3-O-benzyl-2-benzvloxvcarbonylamino-2-deoxv-a-D-
qlucopyranoside (58)
To a solution of compound 57 (20.2 g, 23 mmol) in a 1/1
tetrahydrofuran/methanol mixture (140 ml_) is added, under an inert atmosphere,
[tBu2SnCI(OH)]2 (226 mg, 0.79 mmol) prepared according to A. Orita et al.,
Chem. Eur. J. (2001) 7, 3321. The reaction medium is stirred for 38 hours at
35°C. After concentrating, the residue (20.8 g) is used in the following step
without purification.
Rf = 0.23, silica gel, 1/3 v/v cyclohexane/ethyl acetate
Methyl (methyl 2-O-acetvl-3-O-benzvl-4-O-levulinovl-a-L-
idopvranosyluronate)-(1?4)-3-O-benzvl-2-benzvloxvcarbonylamino-6-O-fe/t-
butyldiphenvlsilyl-2-deoxv-g-D-qlucopvranoside (59)
To a solution of crude compound 58 (23 mmol) in dichloromethane
(190 mL) are added, at 0°C and under an inert atmosphere, triethylamine (8 ml_,
57.5 mmol), 4-dimethylaminopyridine (1.4 g, 11.5 mmol) and tert-
butyldiphenylsilyl chloride (12 mL, 46.0 mmol). The reaction medium is stirred for
16 hours at room temperature. The reaction mixture is diluted with
dichloromethane. The organic phase is washed successively with saturated
aqueous sodium chloride solution and then with water, dried over sodium
sulfate, filtered and then evaporated. The residue is purified by flash
chromatography on a column of silica gel (2/1 cyclohexane/ethyl acetate) to give
compound 59 (24.4 g).
Rf = 0.42, silica gel, 2/1 v/v cyclohexane/ethyl acetate
Methyl (methyl 2-O-acetvl-3-O-benzvl-a-i_-idopvranosyluronate)-(1?4)-3-
0-benzvl-2-benzvloxvcarbonvlamino-6-O-teAt-butvldiphenvlsilvl-2-deoxv-a-D-
qlucopyranoside (60)
To a solution of compound 59 (22.6 g, 20.0 mmol) in a 1/2
toluene/ethanol mixture (2.5 L) is added hydrazine acetate (9.21 g, 100.0 mmol).
The reaction medium is stirred for 30 minutes at room temperature. After
concentrating, the residue is purified by flash chromatography on a column of
silica gel (2/1 cyclohexane/ethyl acetate) to give 17.6 g of compound 60.
Rf = 0.40, silica gel, 2/1 v/v cyclohexane/ethyl acetate
(Methyl 2-O-acetvl-3-O-benzvl-4-O-levulinovl-a-L-idopvranosyluronate)-
(1?4)-1 -O-acetvl-2-azido-3-O-benzyl-2-deoxy-a-P-qlucopvranose (62)
To a solution of 61 (11 g, 13.7 mmol) (prepared according to the method
described by C. Tabeur et ai, Carbohydr. Res., 281 (1996) 253-276) in a 1/1
methanol/tetrahydrofuran mixture (80 ml_) is added [tBu2SnCI(OH)]2 (0.55 g)
prepared according to A. Orita et ai, Chem. Eur. J. (2001) 7, 3321. After stirring
at 35°C for 5.5 hours, and then at room temperature for 16 hours, and then
again at 35°C for 4 hours, the reaction mixture is concentrated under vacuum
and then purified by chromatography to give compound 62 (5.97 g).
LC-MS m/z 780.2 [(M + Na)+]. TR = 9.14 min
(Methyl 2-O-acetvl-3-O-benzvl-4-O-levulinovl-a-L-idopyranosyluronate)-
(1?4)-1-O-acetvl-2-azido-3-O-benzvl-6-O-terf-butvldiphenvlsilvl-2-deoxv-a-D-
qlucopyranose (63)
Compound 62 (5.97 g, 7.88 mmol) is dissolved in dichloromethane
(63 ml_). At 0°C and under argon, 4-dimethylaminopyridine (0.481 g),
triethylamine (2.7 ml_), and ferf-butyldiphenylsilyl chloride (4 ml_) are
successively added. After magnetic stirring for 4 hours, the reaction medium is
diluted with dichloromethane, washed with aqueous 10% potassium hydrogen
sulfate solution, with water, dried (Na2S04), filtered and concentrated. The
residue obtained is purified on silica (ethyl acetate/heptane) to give compound
63 (7 g).
LC-MS m/z 1018.3 [(M + Na)+]. TR = 12.33 min
(Methyl 2-O-acetyl-3-O-benzyl-4-O-levulinovl-a-i_-idopyranosyluronate)-
(1?4)-2-azido-3-O-benzvl-6-O-te/t-butvldiphenylsilvl-2-deoxv-a,|3-D-
glucopyranose (64)
To a solution of compound 63 (7 g, 7.03 mmol) in diethyl ether (70 ml_), is
added, at 0°C, benzylamine (BnNH2) (29 ml_). After stirring for 15 minutes at
0°C, and then for 6 hours at room temperature, the reaction mixture is diluted
with ethyl acetate, and then neutralized with cold 1N HCI, washed with water,
dried (Na2S04), filtered and concentrated, and purified on silica gel (ethyl
acetate/toluene) to give compound 64 (5.86 g).
LC-MS m/z 976.3 [(M + Na)+]. TR = 27.6/27.8 min
(Methyl 2-O-acetvl-3-O-benzvl-4-O-levulinovl-a-L-idopyranosyluronate)-
(1?4)-2-azido-3-O-benzyl-6-O-fe/t-butvldiphenvlsilyl-2-deoxv-a,B-D-
qlucopyranose trichloroacetimidate (65)
To a solution of compound 64 (6.5 g, 6.81 mmol) in dichloromethane
(140 ml_) and in the presence of powdered 4 A molecular sieves (7 g) is added,
under argon, caesium carbonate (CS2CO3) (3.5 g) and then, at 0°C,
trichloroacetonitrile (CCI3CN) (3.4 ml_). After stirring for 15 minutes at 0°C, and
then for 5 hours at room temperature, the reaction mixture is filtered and then
concentrated. The residue is purified on silica gel (1/4 ethyl acetate/toluene +
0.1% triethylamine) to give compound 65 (6.33 g).
LC-MSm/z1119.1 [(M + Na)+]. TR = 31.2
Methyl (methyl 2-O-acetvl-3-O-benzvl-4-O-levulinovl-a-i_-
idopvranosyluronate)-(1?4)-(2-azido-3-Q-benzyl-6-O-teAf-butyldiphenvlsilyl-2-
deoxv-a-D-glucopyranosyl)-(1?4)-(methvl 2-O-acetvl-3-O-benzvl-a-L-
idopvranosyluronate)-(1?4)-3-O-benzvl-2-benzvloxvcarbonvlamino-6-O-fe/f-
butvldiphenvlsilvl-2-deoxv-a-P-glucopvranoside (66)
A mixture of the glycosyl acceptor 59 (8.80 g, 9.00 mmol), the imidate 65
(6.58 g, 6.00 mmol) and powdered 4 A molecular sieves (4.50 g) in
dichloromethane (210 mL) is stirred under an argon atmosphere for 1 hour at
25°C. The reaction mixture is cooled to -20°C and a 1 M solution of tert-
butyldimethylsilyl triflate in dichloromethane (900 uL) is added. After 1 hour
20 minutes, the reaction medium is neutralized by addition of solid sodium
hydrogen carbonate. After filtering through Celite® and concentrating, the
residue obtained is chromatographed on a Sephadex® LH20 column (190 x
3.2 cm, 1/1 dichloromethane/ethanol) to give 8.26 g of compound 66.
Rf = 0.30, silica gel, 2/1 cyclohexane/ethyl acetate
Methyl (methyl 2-O-acetvl-3-O-benzvl-a-L-idopyranosyluronate)-(1?4)-(2-
azido-3-O-benzvl-6-O-te/t-butvldiphenvlsilyl-2-deoxv-a-D-glucopvranosyl)-(1?4)-
(methyl 2-Q-acetyl-3-O-benzvl-a-L-idopyranosyluronate)-(1?4)-3-O-benzyl-2-
benzvloxvcarbonvlamino-6-O-feAt-butvldiphenvlsilyl-2-deoxv-a-D-glucopyranoside
(67)
Compound 66 (8.26 g, 4.31 mmol) is transformed into compound 67
(6.41 g) according to the same procedure as that described for the synthesis of
compound 23.
Rf = 0.34, silica gel, 2/1 cyclohexane/ethyl acetate
Methyl (methyl 2-O-acetvl-3-Q-benzvl-4-O-levulinovl-a-L-
idopvranosyluronate)-(1?4)-(2-azido-3-O-benzvl-6-O-fe/f-butvldiphenvlsilvl-2-
deoxv-a-D-glucopvranosyl(1?4)-(methvl 2-O-acetvl-3-Q-benzvl-a-L-
idopvranosyluronate)-(1?4)-(2-azido-3-O-benzvl-6-O-te/t-butvldiphenvlsilvl-2-
deoxv-a-D-glucopvranosyl)-(1?4Mmethvl 2-O-acetvl-3-Q-benzvl-a-L-
idopvranosyluronate)-f1->4)-3-O-benzvl-2-benzvloxvcarbpnylamino-6-O-te/t-
butvldiphenvlsilyl-2-deoxv-a-D-glucopyranoside (68)
A mixture of the glycosyl acceptor 67 (7.42 g, 4.09 mmol), the imidate 65
(6.73 g, 6.1 mmol), and powdered 4 A molecular sieves (4.60 g) in
dichloromethane (215 mL) is stirred under an argon atmosphere for 1 hour at
25°C. The reaction mixture is cooled to -20°C and a 1 M solution of tert-
butyldimethylsilyl triflate in dichloromethane (920 uL) is added. After 1 hour
30 minutes, the reaction medium is neutralized by addition of solid sodium
hydrogen carbonate. After filtering through Celite®, the reaction medium is
diluted with dichloromethane (800 mL). The organic phase is washed
successively with aqueous 2% sodium hydrogen carbonate solution, with water
and then dried over sodium sulfate, filtered and then evaporated to dryness. The
residue obtained is purified by chromatography on a column of Sephadex® LH20
(190 x 3.2 cm, 1/1 dichloromethane/ethanol) followed by chromatography on a
column of silica gel (6/1 toluene/ethyl acetate) to give 6.13 g of compound 68.
Rf = 0.46, silica gel, 4/1 v/v toluene/ethyl acetate
Methyl (methyl 2-O-acetvl-3-O-benzvl-a-L-idopvranosyluronate)-(1 ->4M2-
azido-3-O-benzvl-6-O-ferf-butvldiphenvlsilyl-2-deoxv-a-p-qlucopyranosyl)- (1?4)-
(methvl 2-O-acetvl-3-O-benzvl-a-L-idopvranosyluronate)-(1?4)-(2-azido-3-Q-
benzvl-6-O-tert-butvldiphenvlsilvl-2-deoxv-a-D-qlucopvranosyl)-(1-^4)-(methvl 2-
0-acetvl-3-O-benzvl-a-L-idopyranosyluronate)-(1?4)-3-O-benzvl-2-
benzvloxvcarbonvlamino-6-O-ferf-butvldiphenvlsilvl-2-deoxv-a-D-qlucopyranoside
(69)
Compound 68 (7.14 g, 2.59 mmol) is transformed into compound 69
(6.07 g) according to the same procedure as that described for the preparation
of compound 23.
Rf = 0.37, silica gel, 2/1 v/v cyclohexane/ethyl acetate
(3.4-di-O-benzvl-2-O-(4-methoxv)benzvl-6-Q-te/f-butvldimethvlsilvl-a-L-
idopvranosv0-(1?4)-1,6-anhvdro-2-azido-3-O-benzvl-2-deoxv-B-D-
qlucopyranose (70)
To a solution of compound 44 (32.3 g, 42.2 mmol) (described in the
preparation of compound 108 of patent application WO 2006/021653) in N,N-
dimethylformamide (210 ml_) is added, at 0°C and under argon, benzyl bromide
(25 ml_) and then 55% NaH (3 g). After 20 minutes of magnetic stirring, methanol
is added (30 mL), the reaction medium is concentrated under vacuum, and the
crude reaction product is diluted with ethyl acetate, washed with water and then
with saturated aqueous sodium chloride solution, dried (Na2SC>4)( filtered and
concentrated. The residue obtained (38.6 g) is used in the following step without
purification.
LC-MS /7i/z871.7 [(M + NH4)+]. TR = 13.86 min
(2-O-acetvl-3.4-di-O-benzvl-6-O-te/t-butvldimethvlsilvl-a-L-idopyranosvn-
(1?4)-1,6-anhvdro-2-azido-3-O-benzvl-2-deoxv-B-D-qlucopyranose (71)
To a solution of crude compound 70 (38.6 g) in dichloromethane (1.6 L)
are added water (80 ml_) and then, at 0°C, DDQ (14.2 g). After stirring for
4 hours 45 minutes at 0°C, the medium is diluted with dichloromethane and
aqueous sodium hydrogen carbonate solution is added. The organic phase is
then washed with water, dried (Na2S04), filtered and concentrated. The
compound obtained is used in the following step without purification.
The residue obtained is dissolved in dichloromethane (350 ml_), and then
triethylamine (13 mL), 4-dimethylaminopyridine (2 g), and acetic anhydride
(60 mL) are added. After magnetic stirring for 10 minutes at 0°C, and then for
1 hour 45 minutes at room temperature, the reaction mixture is diluted with
dichloromethane, and then washed successively with aqueous 10% potassium
hydrogen sulfate solution, water, and the organic phase is then dried (Na2S04),
filtered and concentrated. The residue obtained is purified on silica (ethyl
acetate/cyclohexane) to give compound 71 (26.8 g).
LC-MS m/z 798.3 [(M + Na)*]. TR = 12.97 min
(Methyl 2-O-acetvl-3,4-di-O-benzvl-a-L-idopyranosyluronate)-(1?4)-1,6-
anhvdro-2-azido-3-Q-benzvl-2-deoxv-l3-D-glucopvranose (72)
To a solution of compound 71 (26.3 g, 33.9 mmol) in acetone (1.4 L) is
added, at 0°C, a solution of Cr03 (10.5 g) in aqueous 3.5 M H2S04 (47 mL).
After mechanical stirring for 4 hours at 0°C, the reaction medium is diluted with
dichloromethane, washed with water until neutral, and then the organic phase is
dried (Na2S04), filtered and concentrated. The compound obtained is used in the
following step without purification.
The residue obtained is dissolved in A/,A/-dimethylformamide (210 mL),
and potassium hydrogen carbonate (17 g) and methyl iodide (21 mL) are added.
The reaction mixture is stirred at room temperature for 16 hours, and then
concentrated under vacuum. The residue is diluted with ethyl acetate and then
washed with water, with saturated aqueous sodium thiosulfate solution, with
saturated aqueous sodium chloride solution, and then dried (Na2S04), filtered
and concentrated. The compound obtained is used in the following step without
purification.
LC-MS m/z 707.3 [(M + NH4)+]. TR = 10.37 min
(Methyl 2-O-acetvl-3,4-di-O-benzvl-a-i_-idopvranosyluronate)-(1?4)-1,6-di-
Q-acetvl-2-azido-3-O-benzvl-2-deoxy-a,B-D-qlucopvranose (73)
The crude residue obtained in the preceding step is dissolved in acetic
anhydride (177 mL), and trifluoroacetic acid (TFA) (17.7 mL) is then added. The
reaction mixture is stirred for 16 hours, and is then concentrated, co-evaporated
with toluene, and purified on silica gel (cyclohexane/ethyl acetate), to give
compound 73 (17.4g).
LC-MS m/z 809.3 [(M + NH4)+]. TR = 10.81 min
(Methyl 2-Q-acetvl-3,4-di-O-benzvl-g-L-idopyranosyluronate)-(1?4)-1 -O-
acetvl-2-azido-3-O-benzyl-2-deoxv-a-D-qlucopvranose (74)
To a solution of compound 73 (5.05 g, 6.3 mmol) in a 1/1
methanol/tetrahydrofuran mixture (76 ml_) is added [tBu2SnCI(OH)]2 (0.25 g,
0.88 mmol), prepared according to A. Orita et al., Chem. Eur. J. (2001) 7, 3321.
After stirring at room temperature for 72 hours, the reaction mixture is
concentrated under vacuum and then purified by chromatography to give
compound 74 (2.89 g).
LC-MS m/z 111.4 [(M + Na)4]. TR = 10.23 min
(Methyl 2-O-acetvl-3,4-di-O-benzvl-a-L-idopyranosyluronate)-(1?4)-1-O-
acetvl-2-azido-3-O-benzvl-6-O-terf-butvldiphenvlsilvl-2-deoxv-a-D-qlucopyranose
(75)
Compound 74 (2.89 g, 3.86 mmol) is dissolved in dichloromethane
(31 ml_). At 0°C and under argon, triethylamine (1.3ml_),
4-dimethylaminopyridine (0.235 g) and fe/f-butyldiphenylsilyl chloride (2 mL) are
successively added. After magnetic stirring for 3 hours, the reaction medium is
diluted with dichloromethane, washed with aqueous 10% potassium hydrogen
sulfate solution, with water, dried (Na2S04), filtered and concentrated. The
residue obtained is purified on silica (ethyl acetate/cyclohexane) to give
compound 75 (3.4 g).
LC-MS m/z 1010.6 [(M + Na)+]. TR = 13.10 min
(Methyl 2-O-acetvl-3.4-di-Q-benzvl-a-L-idopvranosyluronate)-(1?4)-2-
azido-3-O-benzvl-6-O-te/f-butyldiphenvlsilvl-2-deoxv-g.B-D-qlucopvranose (76)
To a solution of compound 75 (3.44 g, 3.48 mmol) in diethyl ether (35 mL)
is added, at 0°C, benzylamine (BnNHb) (14.5 mL). After stirring for 8 hours at
room temperature, the reaction mixture is placed at -18°C for 16 hours, and then
again for 2.5 hours at room temperature. The medium is then diluted with ethyl
acetate, and then neutralized with cold 1N HCI, washed with water, dried
(Na2SC>4), filtered and concentrated, and purified on silica gel (ethyl
acetate/cyclohexane 15/85) to give 76 (3.83 g).
LC-MS m/z 963.6 [(M + NH4)1- TR = 12.37, 12.47 min
(Methyl 2-O-acetvl-3,4-di-O-benzvl-a-L-idopvranosyluronateM1->4)-2-
azido-3-O-benzvl-6-O-terf-butvldiphenvlsilvl-2-deoxv-a,p-D-glucopyranose
trichloroacetimidate (77)
To a solution of compound 76 (2.99 g, 3.16 mmol) in dichloromethane
(60 ml_) and in the presence of powdered 4 A molecular sieves (3 g) are added,
at 0°C under argon, caesium carbonate (CS2CO3) (1.6 g), and then
trichloroacetonitrile (CCI3CN) (1.6 mL). After stirring for 20 minutes at 0°C, for
7 hours at room temperature, storing at -18°C for 16 hours, and then magnetic
stirring for 8 hours at room temperature, storing at -18°C for 16 hours, and finally
magnetic stirring for 1 hour at room temperature, the reaction mixture is filtered
and then concentrated. The residue is purified on silica gel (15/85 ethyl
acetate/cyclohexane + 0.1% triethylamine) to give compound 77 (2.69 g).
LC-MS m/z 1113.4 [(M + Na)*]. TR = 14.58 min
Methyl (methyl 2-O-acetvl-314-di-O-benzvl-a-L-idopvranosyluronate)-
(1?4)-(2-azido-3-O-benzvl-6-O-ferf-butvldiphenvlsilvl-2-deoxv-a-D-
glucopvranosylM1->4)-r(methyl 2-O-acetvl-3-O-benzvl-a-i_-idopvranosyluronate)-
(1?4)-(2-azido-3-O-benzvl-6-O-te/t-butvldiphenvlsilyl-2-deoxy-a-D-
glucopyranosyl)-(1?4)12-(methvl 2-O-acetvl-3-O-benzvl-a-L-
idopvranosyluronate)-(1?4)-3-O-benzvl-2-benzvloxvcarbonvlamino-6-O-ferf-
butvldiphenvlsilvl-2-deoxv-a-D-glucopyranoside(78)
A mixture of the glycosyl acceptor 69 (3.50 g, 1.32 mmol), the imidate 77
(2.16 g, 1.98 mmol) and powdered 4 A molecular sieves (1.48 g) in
dichloromethane (69 mL) is stirred under an argon atmosphere for 1 hour at
room temperature. The reaction mixture is cooled to -20°C and a 1 M solution of
te/f-butyldimethylsilyl triflate in dichloromethane (297 uL) is added. After 2 hours
30 minutes, the reaction medium is neutralized by addition of solid sodium
hydrogen carbonate. After filtering through Celite®, the reaction medium is
diluted with dichloromethane (400 mL). The organic phase is washed
successively with aqueous 2% sodium hydrogen carbonate solution, with water
and then dried over sodium sulfate, filtered and then evaporated to dryness. The
residue obtained is purified by chromatography on a column of Sephadex® LH20
(190 x 3.2 cm, 1/1 dichloromethane/ethanol) followed by chromatography on a
column of silica gel (4/1 cyclohexane/ethyl acetate) to give 3.04 g of compound
78.
Rf = 0.30, silica gel, 3/1 cyclohexane/ethyl acetate
Methyl (methyl 3.4-di-O-benzvl-g-L-idopvranosyluronate)-(1 ^4)-(2-azido-
3-O-benzvl-6-O-fe/t-butvldiphenvlsilvl-2-deoxv-a-D-qlucopyranosyl)-(1?4)-
iYmethvl 3-O-benzvl-a-L-idopvranosyluronate)-(1-^4)-(2-azido-3-O-benzvl-6-O-
te/t-butvldiphenylsilvl-2-deoxv-a-D-glucopvranosyl)-(1?4)1?-(methvl 3-O-benzvl-
a-L-idopvranosyluronate)-(1?4)-3-O-benzvl-2-benzvloxvcarbonvlamino-6-O-fe/f-
butvldiphenvlsilyl-2-deoxv-a-D-qlucopvranoside (79)
To a solution of compound 78 (2.23 g, 0.623 mmol) in a 2/3
dichloromethane/methanol mixture (187 mL) containing 3 A molecular sieves
(78 mg) is added, under an argon atmosphere and at 0°C, a 1 M solution of
sodium methoxide in methanol (99.7 uL). After 24 hours at room temperature,
the reaction medium is neutralized with Dowex AG 50 WX4 H+ resin. After
filtering and concentrating, the residue is chromatographed on a column of
Sephadex® LH20 (120 x 3 cm, 1/1 dichloromethane/ethanol) followed by flash
chromatography on a column of silica gel (100/0—>66/34 cyclohexane/ethyl
acetate) to give 1.80 g of compound 79.
Rf = 0.38, silica gel, 3/1 v/v cyclohexane/ethyl acetate
Methyl (methyl 3,4-di-O-benzvl-g-L-idopyranosyluronatey-(1?4)-(2-azido-
3-O-benzvl-6-O-fe/t-butvldiphenvlsilvl-2-deoxv-a-D-qlucopyranosyl)-(1?4)-
[(methyl 3-O-benzvl-g-L-idopvranosyluronate)-(1?4)-(2-azido-3-O-benzvl-6-O-
feAf-butvldiphenvlsilvl-2-deoxv-a-D-glucopyranosyl)-(1-^4)l?-(methvl 3-O-benzyl-
a-L-idopvranosyluronate)-(1-^4)-3-O-benzvl-2-(r(benzyloxv)carbonvnamino>-2-
deoxy-g-D-glucopyranoside (80)
Methyl (methyl 3.4-di-O-benzvl-a-L-idopyranosyluronateMI ->4)-(2-azido-
3-O-benzvl-6-O-feAf-butvldiphenvlsilvl-2-deoxv-a-D-qlucopyranosyl)-(1?4)-
(methvl 3-O-benzvl-g-L-idopvranosyluronate)-(1?4)-(2-azido-3-Q-benzvl-6-O-
teAt-butyldiphenvlsilyl-2-deoxv-g-D-glucopyranosyl)-(1?4)-(methvl 3-O-benzvl-g-
L-idopvranosyluronateM1->4)-(2-azido-3-O-benzvl-2-deoxy-g-D-qlucopyranosyl)-
(1?4)-(methvl 3-O-benzvl-g-L-idopvranosyluronateH1->4)-3-O-benzvl-2-
([(benzvloxv)carbonvnamino)-2-deoxv-g-D-glucopyranoside (81)
Methyl (methyl 3,4-di-Q-benzvl-g-i_-idopvranosyluronate)-(1?4)-(2-azido-
3-Q-benzvl-6-O-ferf-bufyldiphenvlsilyl-2-deoxv-g-D-2-deoxv-g-D-qlucopvranoside(82)
Methyl (methyl 3,4-di-O-benzyl-g-L-idopvranosyluronate)-(1?4)-(2-azido-
3-O-benzvl-2-deoxv-g-D-qlucopyranosyl)-(1?4)-r(methyl 3-O-benzvl-g-L-
idopvranosyluronate)-(1?4)-(2-azido-3-O-benzvl-2-deoxv-g-D-qlucopvranosvn-
(1?4)l£-(methvl 3-O-benzvl-g-L-idopyranosyluronate)-(1?4)-3-Q-benzvl-2-
{r(benzvloxv)carbonvnamino>-2-deoxv-g-D-glucopyranoside (87)
To a solution of compound 79 (373 mg, 0.11 mmol) in methanol (14 ml_)
is added ammonium fluoride (324 mg, 8.74 mmol). After stirring for 20 hours at
room temperature, the reaction mixture is deposited on a column of Sephadex®
LH20 gel (120 x 3 cm, 1/1 v/v dichloromethane/ethanol) followed by flash
chromatography on a column of silica gel (1/0-+7/3 v/v toluene/acetone) to give
successively:
- Compound 80 (58.8 mg)
TLC: Rf = 0.63, silica gel, 65/35 v/v toluene/acetone
- Compound 81 (44.4 mg)
TLC: Rf = 0.53, silica gel, 65/35 v/v toluene/acetone
- Compound 82 (37.7 mg)
TLC: Rf = 0.45, silica gel, 65/35 v/v toluene/acetone
- A mixture of compound 83 and of compound 84 (54.0 mg)
- A mixture of compound 85 and of compound 86 (48.3 mg)
- Compound 87 (26.6 mg)
TLC: Rf = 0.14, silica gel, 65/35 v/v toluene/acetone
Methyl (methyl 3,4-di-O-benzvl-g-L-idopvranosyluronateH1 ->4H2-azido-
3-Q-benzvl-2-deoxv-a-D-qlucopvranosylM1->4Mmethvl________3-O-benzyl-g-L-
idopyranosyluronate)-(1?4)-(2-azido-3-O-benzvl-6-O-fe/t-butvldiphenvlsilyl-2-
deoxy-g-D-qlucopvranosyl)-(1?4)-(methvl 3-O-benzyl-a-L-idopvranosyluronate)-
(1?4)-(2-azido-3-O-benzvl-6-O-fe/f-butyldiphenvlsilvl-2-deoxv-a-D-
qlucopvranosyl)-(1?4)-(methyl 3-O-benzvl-g-L-idopvranosyluronate)-(1?4)-3-Q-
benzyl-2-(r(benzvloxv)carbonvnamino>-2-deoxv-a-D-qlucopyranoside (83)
Methyl (methyl 3.4-di-O-benzvl-a-L-idopvranosyluronate)-(1?4)-(2-azido-
3-O-benzvl-6-O-te/f-butvldiphenvlsilvl-2-deoxv-a-D-qlucopyranosyl)-(1?4)-
lYmethyl 3-O-benzvl-g-L-idopvranosyluronate)-(1?4)-(2-azido-3-O-benzvl-2-
deoxv-g-D-qlucopvranosyl)-(1?4)1?-(methyl 3-O-benzvl-g-L-idopvranosyluronate)-
(1?4)-3-O-benzvl-2-(f(benzvloxv)carbonvnamino>-2-deoxv-g-D-glucopyranoside
(84)
The mixture of compounds 83 and 84 (260 mg) is purified by flash
chromatography on a column of silica gel (100/0-^85/15 v/v toluene/methanol) to
give compound 83 (27.8 mg).
TLC: Rf =0.22, silica gel, 85/15 v/v toluene/methanol
The remaining fractions are repurified by flash chromatography on a
column of silica gel (100/0-^97/3 v/v dichloromethane/methanol) to give
compound 84 (45.3 mg).
TLC: Rf = 0.13, silica gel, 85/15 v/v toluene/methanol
Methyl (methyl 3,4-di-O-benzvl-a-L-idopvranosyluronate)-(1?4)-(2-azido-
3-O-benzvl-2-deoxv-g-D-glucopvranosyl)-(1?4)-(methyl 3-O-benzvl-a-L-
idopvranosyluronate)-(1?4)-(2-azido-3-O-benzvl-6-O-te/f-butvldiphenylsilvl-2-
deoxv-g-D-glucopvranosyl)-(1?4)-(methvl 3-O-benzvl-a-L-idopyranosyluronate)-
(,1^4)-(2-azido-3-O-benzyl-2-deoxy-a-D-glucopvranosyl)-n->4)-(methvl 3-O-
benzvl-a-L-idopyranosyluronate)-(1-^4)-3-O-benzyl-2-{T(benzvloxv)carbonyn-
amino|-2-deoxy-a-D-glucopvranoside (85)
Methyl (methyl 3,4-di-O-benzvl-g-i_-idopvranosyluronate)-(1?4)-(2-azido-
3-O-benzyl-2-deoxy-g-D-glucopvranosyl)-(1?4)-(methyl 3-O-benzyl-a-L-
idopyranosylurpnate)-(1?4)-(2-azido-3-O-benzvl-2-deoxv-a-D-glucopyranosyl)-
(1?4)-(methvl 3-O-benzvl-a-L-idopyranosyluronate)-(1?4)-(2-azido-3-Q-benzyl-
6-O-feAf-butyldiphenvlsilyl-2-deoxy-a-D-glucopyranosyl)-(1-^4)-(methvl 3-Q-
benzyl-a-L-idopyranosyluronate)-(1?4)-3-O-benzyl-2-(f(benzvloxv)carbonyn-
amino}-2-deoxv-a-D-glucopyranoside (86)
The mixture of compounds 85 and 86 (135 mg) is purified by HPLC
chromatography on a column of C18 silica gel (Waters® Sunfire, 5 urn, 150 x
19 mm, 9/1 acetonitrile/water + 0.1% trifluoroacetic acid) to give successively:
- Compound 85 (32.6 mg), after size exclusion chromatography
(Sephadex® LH20, 120 x 3 cm, 1/1 v/v dichloromethane/ethanol)
Mass: "ESI" method, negative mode: theoretical mass = 2698.96;
experimental mass: 2698.55 ± 0.65 a.m.u.
- Compound 86 (26.5 mg), after size exclusion chromatography
(Sephadex® LH20, 120 x 3 cm, 1/1 v/v dichloromethane/ethanol)
Mass: "ESI" method, negative mode: theoretical mass = 2698.96;
experimental mass: 2698.95 ± 1.05 a.m.u.
Methyl (methyl 3,4-di-O-benzvl-2-O-triethylammonium sulfonato-g-L-
idopvranosyluronate)-(1?4)-(2-azido-3-O-benzvl-6-Q-te/t-butyldiphenvlsilvl-2-
deoxv-a-D-qlucopvranosyl)-(1?4)-(methvl 3-Q-benzvl-2-O-triethvlammonium
sulfonato-a-L-idopvranosyluronate)-(1?4)-(2-azido-3-O-benzvl-6-O-fe/t-butyl-
diphenvlsi[vl-2-deoxv-a-D-glucopyranosyl)-(1?4)-(methvl_______3-O-benzyl-2-Q-
triethylammonium______sulfonatOKx-L-idopyranosyluronateH1->4)-(2-azido-3-O-
benzyl-2-deoxv-6-O-triethylammonium sulfonato-a-D-glucopyranosyl)-(1?4)-
(methyl 3-O-benzvl-2-O-triethylammonium sulfonato-a-L-idopyranosyluronate)-
(1?4)-3-O-benzvl-2-(r(benzvloxv)carbonvnamino>-2-deoxv-6-O-
triethylammonium sulfonato-a-D-glucopyranoside (88)
Compound 81 (69 mg, 23.5 umol) is treated according to the same
procedure as that described for the preparation of compound 28 to give
compound 88 (85.1 mg) after size exclusion chromatography (Sephadex® LH20,
120 x 3 cm, 1/1 v/v dichloromethane/ethanol).
TLC: Rf = 0.62, silica gel, 17/9/2.2/5 v/v/v/v ethyl acetate/pyridine/acetic
acid/water.
Methyl (methyl 3,4-di-O-benzvl-2-Q-ammonium sulfonato-a-L-
idopvranosyluronate)-(1->4M2-azido-3-O-benzvl-2-deoxv-g-D-qlucopvranosyl)-
(1 ->4Mmethvl 3-O-benzvl-2-O-ammonium suifonato-g-L-idopvranosyluronate)-
(1?4)-(2-azido-3-O-benzvl-2-deoxv-g-D-olucopyranosv0-(1?4)-(methvl 3-O-
benzyl-2-O-ammonium sulfonato-g-L-idopvranosyluronate)-(1->4M2-azido-3-O-
benzvl-2-deoxv-6-O-ammonium sulfonato-g-D-qlucopvranosyl)-(1 ->4Mmethvl 3-
O-benzyl-2-O-ammonium sulfonato-g-L-idopyranosyluronateH1->4)-3-Q-benzyl-
2-([(benzvloxv)carbonynamino}-2-deoxv-6-O-ammonium sulfonato-g-D-
qlucopyranoside (89)
To a solution of compound 88 (85.1 mg, 21.1 umol) in methanol (2.7 ml_)
is added ammonium fluoride (31.3 mg, 0.846 mmol). After 48 hours at 55°C, the
reaction mixture is deposited on a column of Sephadex® LH20 gel (95 x 2 cm)
eluted with A/,A/-dimethylformamide to give compound 89 (64 mg).
[a]D 19.4° (c 1.0; MeOH)
Methyl (lithium 3.4-di-Q-benzvl-2-O-lithium sulfonato-a-L-
idopvranosyluronate)-(1?4)-(2-azido-3-O-benzvl-2-deoxv-a-D-qlucopvranosyl)-
(1?4)-(lithium 3-Q-benzyl-2-Q-lithium sulfonato-a-L-idopyranosyluronate)-(1 ->4V
(2-azido-3-O-benzvl-2-deoxv-a-D-qlucopvranosyl)-(1?4)-(lithium 3-Q-benzyl-2-
O-lithium sulfonato-a-L-idopvranosyluronate)-(1-^4)-(2-azido-3-O-benzvl-2-
deoxy-6-O-lithium sulfonato-a-D-qlucopvranosylM1 -^4)-(lithium 3-O-benzyl-2-Q-
lithium sulfonato-a-L-idopyranosyluronate)-(1?4)-3-Q-benzvl-2-
(r(,benzvloxv)carbonvnamino>-2-deoxv-6-O-lithium sulfonato-a-D-qlucopyranoside
(90)
Compound 89 (63.3 mg; 17.8 umol) is treated according to the same
procedure as that described for the preparation of compound 29.
Chromatography on a column of Sephadex® LH20 gel (120 x 3 cm, 50/50/1 v/v/v
dichloromethane/ethanol/water) gives compound 90 (49.8 mg).
[a]D 14.6° (c 1.0; MeOH)
Methyl (sodium 2-O-sodium sulfonato-a-L-idopvranosyluronateM1->4M2-
amino-2-deoxv-a-D-glucopyranosylH1 ->4Msodium 2-Q-sodium sulfonato-a-L-
idopvranosyluronateMW4H2-amino-2-deoxy-a-D-glucopyranosylM1->4)-
(sodium 2-O-sodium suifonato-a-i_-idopvranosyluronateM1 ->4M2-amino-2-
deoxy-6-O-sodium sulfonato-a-D-glucopvranosyl)-(1 ->4Hsodium 2-O-sodium
sulfonato-a-L-idopvranosyluronate)-(1?4)-2-amino-2-deoxy-6-O-sodium
sulfonato-a-D-qlucopyranoside (91)
To a solution of compound 90 (7.6 mg; 2.6 umol) in a 1/1 v/v tert-
butanol/water mixture (516 uL) are successively added, at room temperature,
ammonium formate (21.2 mg; 0.33 umol) and 10% palladium-on-charcoal
(49.4 mg). After stirring for 4 hours, the reaction medium is filtered (Millipore®
LSWP 5 urn filter) and deposited on a column of Sephadex® G25-fine gel (95 x
2 cm) eluted with aqueous 0.2 M NaCI solution. The fractions containing the
expected compound are combined and deposited on a column of Sephadex®
G25-fine gel (95 x 2 cm) eluted with water. The crude product 91 thus obtained
(5.5 mg) is used without further purification in the following step.
Methyl (methyl 3.4-di-O-benzvl-2-O-triethylammonium sulfonato-a-L-
idopvranosyluronate)-(1?4)-(2-azido-3-O-benzvl-6-O-fe/f-butvldiphenvlsilvl-2-
deoxv-a-D-glucopvranosylM1->4Mmethyl 3-Q-benzvl-2-O-triethylammonium
sulfonato-a-L-idopvranosyluronate)-(1->4H2-azido-3-O-benzvl-2-deoxv-6-Q-
triethvlammonium sulfonato-a-D-qlucopyranosyl)-(1?4)-(methvl 3-Q-benzvl-2-Q-
triethvlammonium sulfonato-a-L-idopvranosyluronateM1->4)-(2-azido-3-Q-
benzvl-2-deoxv-6-O-ferf-butvldiphenvlsilvl-a-D-qlucopvranosyl)-(1^4Mmethvl 3-
O-benzvl-2-O-triethvlammonium sulfonato-a-L-idopvranosyluronate)-(1?4)-3-O-
benzvl-2-{r(benzvloxv)carbonvnamino)-2-deoxv-6-O-triethylammonium sulfonato-
g-p-glucopyranoside (92)
Compound 82 (16.5 mg, 5.6 umol) is treated according to the same
procedure as that described for the preparation of compound 28 to give
compound 92 (21.5 mg) after chromatography on a column of Sephadex® LH20
gel (120 x 3 cm, 1/1 v/v dichloromethane/ethanol).
TLC: Rf = 0.73, silica gel, 28/16/3.8/9 v/v/v/v ethyl acetate/pyridine/acetic
acid/water.
Methyl (methyl 2-O-ammonium sulfonato-3,4-di-Q-benzvl-g-L-
idopvranosyluronate)-(1?4)-(2-azido-3-O-benzvl-2-deoxv-a-D-qlucopvranosyl)-
(1?4)-(methvl 2-O-ammonium sulfonato-3-O-benzvl-a-L-idopvranosyluronate)-
(1?4)-(6-O-ammonium sulfonato 2-azido-3-O-benzvl-2-deoxv-a-D-
qlucopvranosyl)-(1?4)-(methvl 2-O-ammonium sulfonato-3-O-benzvl-g-L-
idopvranosyluronate)-(1-^4)-(2-azido-3-O-benzvl-2-deoxv-a-D-qlucopyranosyl)-
(1?4)-(methvl 2-O-ammonium sulfonato-3-O-benzvl-a-L-idopyranosyluronate)-
(1?4)-6-Q-ammonium sulfonato-3-O-benzvl-2-(r(benzvloxy)carbonvnamino>-2-
deoxv-g-D-qlucopyranoside (93)
Compound 92 (21.5 mg, 5.3 umol) is treated according to the same
procedure as that described for the preparation of compound 89 to give
compound 93 (21.5 mg) after size exclusion chromatography (Sephadex® LH20,
95 x 2 cm, A/,A/-dimethylformamide).
TLC: Rf = 0.63, silica gel, 11/7/1.6/4 v/v/v/v ethyl acetate/pyridine/acetic
acid/water.
Methyl (lithium 3,4-di-O-benzvl-2-O-lithium sulfonato-a-L-
idopvranosyluronate)-(1?4)-(2-azido-3-O-benzvl-2-deoxv-g-D-alucopvranosyl)-
(1?4)-(lithium 3-Q-benzvl-2-Q-lithium sulfonato-g-L-idopvranosyluronate)-(1?4)-
(2-azido-3-Q-benzvl-2-deoxv-6-O-lithium sulfonato-g-D-qlucopvranosvO-(1?4)-
(lithium 3-O-benzyl-2-O-lithium sulfonato-a-L-idopyranosyluronateVd ->4)-(2-
azido-3-O-benzvl-2-deoxv-a-D-glucopyranosyl)-(1?4)-(lithium 3-Q-benzvl-2-O-
lithium sulfonato-g-i_-idopvranosyluronate)-(1?4)-3-O-benzvl-2-
(f(benzvloxv)carbonyl1amino)-2-deoxv-6-O-lithium sulfonato-a-p-glucopyranoside
(94)
Compound 93 (14.7 mg, 4.1 umol) is treated according to the same
procedure as that described for the preparation of compound 31 to give
compound 94 (13.0 mg) after size exclusion chromatography (Sephadex® LH20,
95 x 2 cm, 50/50/1 v/v/v dichloromethane/ethanol/water).
TLC: Rf = 0.56, silica gel, 28/16/3.8/9 v/v/v/v ethyl acetate/pyridine/acetic
acid/water
Methyl (sodium 2-O-sodium sulfonato-g-L-idopvranosyluronate)-(1?4)-(2-
amino-2-deoxv-a-D-qlucopvranosyl)-(1?4)-(sodium 2-O-sodium sulfonato-a-L-
idopvranosyluronate)-(1?4)-(2-amino-2-deoxv-6-O-sodium sulfonato-g-D-
glucopyranosvD-d ->4)-(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-
(1 -^¦4)-(2-amino-2-deoxy-g-D-glucopvranosyl)-(1?4)-(sodium 2-O-sodium
sulfonato-a-L-idopyranosyluronate)-(1?4)-2-amino-2-deoxv-6-O-sodium
sulfonato-a-D-qlucopyranoside (95)
To a solution of compound 94 (13 mg, 4.4 umol) in a 1/1 v/v tert-
butanol/water mixture (883 uL) are added ammonium formate (36 mg,
0.57 mmol) and 10% palladium-on-charcoal (34 mg). After stirring for 4 hours at
room temperature, the reaction medium is filtered (Millipore® LSWP 5 urn filter)
and concentrated to dryness. The residue is deposited on a column of
Sephadex® G25-fine gel (95 x 2 cm) eluted with aqueous 0.2 M NaCI solution.
The fractions containing the expected compound are combined and deposited
on a column of Sephadex® G25-fine gel (95 x 2 cm) eluted with water. The crude
product 95 thus obtained (2.5 mg) is used without further purification in the
following step.
SCHEME 20: preparation of the octasaccharide 99
Methyl (methyl 3,4-di-O-benzvl-2-O-triethvlammonium sulfonato-a-L-
idopvranosyluronate)-(1-^4)-(2-azido-3-O-benzvl-2-deoxv-6-O-triethylammonium
sulfonato-a-D-glucopvranosylM1->4H(methvl 3-O-benzyl-2-O-triethvlammonium
sulfonato-a-L-idopvranosyluronate)-(1?4)-(2-azido-3-O-benzvl-6-Q-te/f-
butyldiphenvlsilvl-2-deoxv-a-D-qlucopyranosyl)-(1?4)-(methyl 3-Q-benzyl-2-Q-
triethvlammonium sulfonato-a-i_-idopyranosyluronateM1->4)lg-3-O-benzvl-2-
(f(benzvloxvkarbonvnamino)-2-deoxv-6-O-triethvlammoriiumsulfonato-a-D-
qlucopyranoside (96)
Compound 83 (35.1 mg , 12.0 umol) is treated according to the same
procedure as that described for the preparation of compound 28 to give
compound 96 (46.3 mg) after chromatography on a column of Sephadex® LH20
gel (120 x 3 cm, 1/1 v/v dichloromethane/ethanol).
[a]D 111° (c 0.95; CH2CI2)
Methyl (methyl 2-O-ammonium sulfonato-3,4-di-O-benzyl-a-L-
idopyranosyluronate)-(1?4)-(6-Q-ammonium sulfonato-azido-3-O-benzyl-2-
deoxv-a-D-glucopyranosyl)-(1?4)-(methyl 2-O-ammonium sulfonato 3-Q-benzyl-
a-L-idopvranosyluronate)-(1?4)-(2-azido-3-O-benzyl-2-deoxv-a-D-
glucopyranosyl)-(1?4)-(methyl 2-O-ammonium sulfonato-3-O-benzyl-a-L-
idopyranosyluronate)-(1?4)-(2-azido-3-O-benzvl-2-deoxv-a-D-qlucopyranosyl)-
(1 ->4Mmethvl 2-O-ammonium sulfonato-3-O-benzyl-a-L-idopvranosyluronate)-
(1?4)-6-Q-ammonium sulfonato-3-O-benzvl-2-(f(benzyloxv)carbonvnamino}-2-
deoxv-g-D-qlucopyranoside (97)
Compound 96 (45.0 mg, 11.2 umol) is treated according to the same
procedure as that described for the preparation of compound 89 to give
compound 97 (30.8 mg) after chromatography on a column of Sephadex® LH20
gel (95 x 2 cm, A/,A/-dimethylformamide).
[4Vf2-azido-3-O-benzvl-2-deoxv-6-Q-lithium sulfonato-
a-D-qlucopvranosyl)-(1 -»4M(lithium 3-Q-benzyl-2-O-lithium sulfonato-a-L-
idopvranosyluronateM1^4H2-azido-3-O-benzyl-2-deoxv-a-D-qlucopyranosyl)-
(1->4YI?-(lithium methyl 3-O-benzvl-2-Q-lithium sulfonato-a-L-
idopvranosyluronate)-(1?4)-3-O-benzvl-2-([(benzyloxv)carbonvnamino)-2-
deoxy-6-O-lithium sulfonato-a-D-qlucopyranoside (98)
Compound 97 (27.1 mg, 7.6 umol) is treated according to the same
procedure as that described for the preparation of compound 29 to give
compound 98 (20.7 mg) after chromatography on a column of Sephadex® LH20
gel (95 x 2 cm, 50/50/1 v/v/v dichloromethane/ethanol/water).
[a]D 14.4° (c 1.01; MeOH)
Methyl (sodium 2-O-sodium sulfonato-a-L-idopvranosyluronateM1—>4)-(2-
amino-2-deoxv-6-O-sodium sulfonato-a-D-glucopyranosyl)-(1?4)-f(sodium 2-O-
sodium sulfonato-a-L-idopvranosyluronate)-(1?4)-(2-amino-2-deoxv-a-D-
glucopvranosylM1->4)l?-(sodium 2-Q-sodium sulfonato-ct-L-
idopvranosyluronateM1->4)-2-amino-2-deoxv-6-O-sodium sulfonato-a-D-
glucopyranoside (99)
Compound 98 (17.5 mg, 5.9 umol) is treated according to the same
procedure as that described for the preparation of compound 91 to give
compound 99 (9.5 mg).
Mass: "ESI" method, negative mode: theoretical mass = 2081.38;
experimental mass: 1993.28 + 0.14 a.m.u. (iduronic acids observed in COOH
form).
Methyl (methyl 3.4-di-O-benzvl-g-L-idopyranosyluronateM1 ->4)-(2-azido-
3-O-benzvl-6-O-fe/f-butvldiphenvlsilvl-2-deoxv-g-D-qlucopyranosv0-(1?4)-
lYmethyl 3-O-benzvl-g-L-idopvranosyluronateH1->4M2-azido-3-O-benzvl-6-O-
fe/f-butvldiphenvlsilvl-2-deoxv-a-D-glucopvranosyl)-(1-^4)l7-(methvl 3-O-benzvl-
a-L-idopvranosyluronateV(1?4)-6-Q-benzovl-3-O-benzvl-2-{f(benzvloxv)-
carbonvl1aminoV2-deoxv-a-D-qlucopvranoside (100)
A solution of compound 80 (355 mg , 0.117 mmol), benzoic anhydride
(75.8 mg, 0.335 mmol) and triethylamine (47.3 uL, 0.335 mmol) in
1,2-dichloroethane (5.6 ml_) is stirred at 60°C for 24 hours and then for 64 hours
at room temperature. The reaction mixture is deposited on a column of
Sephadex® LH20 gel (120 x 3 cm) eluted with a 1/1 v/v dichloromethane/ethanol
mixture, followed by flash chromatography on a column of silica gel
(100/0-^-85/15 v/v toluene/acetone) to give compound 100 (193.2 mg).
TLC: Rf = 0.38, silica gel, 2/1 v/v cyclohexane/ethyl acetate
Methyl (methyl 3,4-di-Q-benzvl-a-L-idopvranosyluronateM1 ->4)-(2-azido-
3-O-benzvl-2-deoxv-a-D-qlucopvranosylH1->4)-F(methyl 3-O-benzyl-a-L-
idopvranosyluronate)-(1?4)-(2-azido-3-O-benzvl-2-deoxv-a-D-qlucopyranosyl)-
(1?4)l?-(methvl 3-O-benzvl-a-L-idopyranosyluronate)-(1?4)-6-Q-benzoyl 3-O-
benzvl-2-(f(benzvloxv)carbonvllamino)-2-deoxy-a-D-qlucopyranoside (101)
Compound 100 (60.0 mg, 0.018 mmol) is treated according to the same
procedure as that described for the preparation of compound 89 to give
compound 101 (46.1 mg) after chromatography on a column of Sephadex®
LH20 gel (120 x 3 cm, 1/1 v/v dichloromethane/ethanol).
Mass: "ESI" method, negative mode: theoretical mass = 2564.66;
experimental mass: 2563.66 ± 0.19 a.m.u.
Methyl (methyl 3.4-di-O-benzvl-2-O-triethvlammonium sulfonato-g-L-
idopvranosyluronate)-(1-^4)-(2-azido-3-O-benzyl-2-deoxv-6-O-triethvlammonium
sulfonato-g-D-qlucopyranosyl)-(1?4)-r(methvl 3-Q-benzyl-2-O-triethvlammonium
sulfonato-g-L-idopvranosyluronate)-(1?4)-(2-azido-3-O-benzvl-2-deoxy-6-O-
triethylammonium sulfonato-g-D-glucopvranosyl)-(1?4)1?-(methyl 3-Q-benzvl-2-
O-triethvlammonium sulfonato-g-i_-idopvranosyluronate)-(1?4)-6-Q-benzovl-3-O-
benzvl-2-(f(benzvloxv)carbonvnamino)-2-deoxv-a-D-glucopyranoside (102)
Compound 101 (42.9 mg, 0.016 mmol) is treated according to the same
procedure as that described for the preparation of compound 28 to give
compound 102 (28.7 mg) after chromatography on a column of Sephadex®
LH20 gel (120 x 3 cm, 1/1 v/v dichloromethane/ethanol) and then flash
chromatography on a column of C-i8 silica gel (40-60 urn; A: methanol, 5% water,
23 mM ammonium acetate; B: acetonitrile, 45% methanol, 5% water, 17 mM
ammonium acetate; A -*• B 100/0^-70/30) followed by desalting on a Sephadex®
LH20 column (95 x 2 cm, 50/50/1 v/v/v dichloromethane/ethanol/water).
Methyl (lithium 3,4-di-O-benzvl-2-O-lithium sulfonato-g-L-
idopvranosyluronate)-(1?4)-(2-azido-3-Q-benzvl-2-deoxv-6-Q-lithium sulfonato-
g-D-glucopvranosyl)-(1?4)-f(lithium 3-Q-benzyl-2-O-lithium sulfonato-a-L-
idopvranosyluronate)-(1?4)-(2-azido-3-Q-benzvl-2-deoxv-6-O-lithium sulfonato-
g-D-glucopyranosyl)-(1?4)l;>-(lithium 3-O-benzvl-2-Q-lithium sulfonato-g-L-
idopvranosyluronate)-(1-^4)-3-Q-benzvl-2-{T(benzyloxv)carbonvnaminoV2-
deoxy-g-D-glucopyranoside (103)
Compound 102 (28.7 mg, 7.5 umol) is treated according to the same
procedure as that described for the preparation of compound 29 to give crude
compound 103 (17.3 mg).
Methyl (sodium 2-O-sodium sulfonato-ti-L-idopvranosyluronate)-(1—>4)-(2-
amino-2-deoxv-6-O-sodium sulfonato-g-D-glucopvranosyl)-(1 ->4H(sodium 2-O-
sodium sulfonato-g-L-idopyranosyluronate)-(1?4)-(2-amino-2-deoxv-6-O-sodium
sulfonato-a-D-glucopyranosvD-d ->4)1r(sodium 2-O-sodium sulfonato-g-L-
idopvranosyluronate)-(1-*4)-2-amino-2-deoxv-g-D-glucopyranoside (104)
Compound 103 (17.3 mg, 5.7 umol) is treated according to the same
procedure as that described for the preparation of compound 91. The crude
compound 104 thus obtained is again saponified. To a solution of crude
compound 104 in a 1/1 v/v tetrahydrofuran/methanol mixture (506 uL) is added,
at 0°C, a 0.7 M solution of lithium hydroxide in water (202 uL, qs a final
concentration of 0.2 M). After 1 hour at 0°C and then 16 hours at room
temperature, the reaction medium is chromatographed on an exclusion column
(Sephadex® G25-fine, 95 x 2 cm, 0.2 M NaCI and then Sephadex® G25-fine, 95
x 2 cm, water) to give compound 104 (6.5 mg).
1H NMR [600 MHz] (D20) 5 of the anomeric protons: 5.45; 5.44; 5.43;
5.26; 5.25; 5.24; 5.18; 4.98 ppm
Methyl (methyl 3,4-di-O-benzyl-2-O-triethvlammonium sulfonato-g-L-
idopvranosyluronate)-n-»4)-(2-azido-3-O-benzvl-2-deoxv-6-O-triethvlammonium
sulfonato-a-D-glucopyranosylM1-^4Mmethyl 3-Q-benzvl-2-O-triethylammonium
sulfonato-a-L-idopvranosyluronate)-(1?4)-(2-azido-3-Q-benzvl-6-O-fe/t-
butvldiphenvlsilvle-2-deoxv-a-D-glucopvranosyl)-(1?4)-(methvl 3-Q-benzyl-2-Q-
triethylammonium sulfonato-g-L-idopvranosyluronateH1->4M2-azido-3-Q-
benzvl-2-deoxv-6-O-triethylammonium sulfonato-g-D-qlucopvranosylM1->4)-
fmethvl 3-O-benzvl-2-O-triethylammonium sulfonato-g-L-idopyranosyluronate)-
(1-^4)-3-O-benzvl-2-deoxv-2-f(benzvloxy)carbonvnamino-6-O-triethylammonium
sulfonato-g-D-qlucopyranoside (105)
Compound 85 (31.4 mg, 11.6 umol) is treated according to the same
procedure as that described for the preparation of compound 28 to give
compound 105 (40.2 mg) after chromatography on a column of Sephadex®
LH20 gel (120 x 3 cm, 50/50/1 v/v/v dichloromethane/ethanol/water).
Methyl (methyl 2-O-ammonium sulfonato-3,4-di-Q-benzvl-a-L-
idopvranosyluronateM1->4)-(6-Q-ammonium sulfonato-2-azido-3-Q-benzyl-2-
deoxv-g-D-glucopyranosvO-d ->4)-(methyl 2-O-ammonium sulfonato-3-O-benzyl-
g-L-idopvranosyluronate)-(1->4M2-azido-3-Q-benzyl-2-deoxv-g-D-
qlucopyranosyl)-(1?4)-(methvl 2-O-ammonium sulfonato-3-O-benzyl-a-L-
idopyranosyluronate)-(1?4)-(6-O-ammonium sulfonato-2-azido-3-Q-benzvl-2-
deoxv-a-D-qlucopvranosyl)-(1 -44)-(methyl 2-O-ammonium sulfonato-3-O-benzvl-
a-L-idopyranosyluronate)-(1?4)-6-O-ammonium sulfonato-3-O-benzyl-2-
f(benzvloxv)carbonvnamino-2-deoxv-g-D-qlucopyranoside (106)
Compound 105 (38.4 mg, 9.68 umol) is treated according to the same
procedure as that described for the preparation of compound 89 to give
compound 106 (28.4 mg) after chromatography on a column of Sephadex®
LH20 gel (95 x 2 cm, N,A/-dimethylformamide).
Rf = 0.21 (28/16/3.8/9 EtOAc/pyridine/AcOH/H20).
Methyl (lithium 3,4-di-O-benzvl-2-Q-lithium sulfonato-g-L-
idopvranosyluronate)-(1?4)-(2-azido-3-O-benzyl-2-deoxv-6-O-lithium sulfonato-
g-D-glucopvranosylM1 -»4Mlithium 3-O-benzvl-2-O-lithium sulfonato-g-L-
idopvranosyluronate)-(1?4)-(2-azido-3-O-benzvl-2-deoxv-g-D-qlucopyranosyl)-
(1?4)-(lithium 3-Q-benzvl-2-Q-lithium sulfonato-g-L-idopyranosyluronate)-(1 -»4V
(2-azido-3-O-benzyl-2-deoxv-6-O-lithium sulfonato-g-D-qlucopyranosyl)-(1?4)-
(lithium 3-O-benzvl-2-O-lithium sulfonato-a-L-idopvranosyluronate)-(1 ^4)-3-Q-
benzvl-2-deoxv-2-r(benzyloxv)carbonvnamino-6-O-lithium sulfonato-a-D-
qlucopyranoside (107)
Compound 106 (28.4 mg, 7.62 umol) is treated according to the same
procedure as that described for the preparation of compound 89 to give
compound 107 (28.0 mg) after chromatography on a column of Sephadex®
LH20 gel (95 x 2 cm, 50/50/1 v/v/v dichloromethane/ethanol/water).
Rf = 0.12 (28/16/3.8/9 EtOAc/pyridine/AcOH/H20).
Methyl (sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1-4)-(2-
amino-2-deoxv-6-O-sodium sulfonato-a-D-qlucopvranosylM1 -4)-(sodium 2-O-
sodium sulfonato-a-L-idopvranosyluronate)-(1-4)-(2-amino-2-deoxv-a-D-
qlucopvranosyl)-(1-4)-(sodium 2-O-sodium sulfonato-a-L-idopvranosyluronate)-
(1 -4)-(2-amino-2-deoxv-6-O-sodium sulfonato-a-D-qlucopyranosyl)-(1 -4)-(sodium
2-O-sodium sulfonato-a-L-idopvranosyluronate)-(1-4)-2-amino-2-deoxv-6-O-
sodium sulfonato-g-D-qlucopyranoside (108)
Compound 107 (5.8 mg, 2.85 umol) is treated according to the same
procedure as that described for the preparation of compound 91 to give
compound 108 (2.5 mg), which is used without further purification in the
following step.
1H NMR [600 MHz] (D20) 6 of the anomeric protons: 5.45; 5.44; 5.42;
5.26; 5.25; 5.24; 5.17; 5.03-ppm
Methyl (methyl 3,4-di-O-benzvl-2-O-triethylammonium sulfonato-g-L-
idopvranosyluronate)-(1?4)-(2-azido-3-O-benzvl-2-deoxv-6-O-triethylammonium
sulfonato-a-D-qlucopyranosyl)-f(1?4)-(methyl 3-Q-benzvl-2-O-triethylammonium
sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-azido-3-O-benzyl-2-deoxv-6-O-
triethvlammonium sulfonato-a-p-glucopyranosyl)1?-(1?4)-(methvl 3-Q-benzyl-2-
Q-triethylammonium sulfonato-a-L-idopyranosyluronate)-(1?4)-3-O-benzvl-2-
benzvloxvcarbonvlamino-2-deoxv-6-O-triethylammonium sulfonato-a-D-
qlucopyranoside (109)
Compound 87 (37.0 mg, 0.15 mmol) is dried by co-distillation of N,N-
dimethylformamide (3 x 1.4 mL) and is then dissolved in A/,A/-dimethylformamide
(1.4mL). To this solution is added the sulfur trioxide-triethylamine complex
(109 mg; 6.01 mmol). The mixture is stirred for 16 hours at 55°C protected from
light and the excess reagent is then destroyed with methanol (25 jjL). The
reaction medium is deposited on a column of Sephadex® LH20 (120 x 3 cm)
eluted with a 1/1 dichloromethane/ethanol mixture to give compound
109 (59.8 mg).
Rf = 0.26, silica gel, 28/16/3.8/9 ethyl acetate/pyridine/acetic acid/water.
Methyl (lithium 3,4-di-O-benzyl-2-O-lithium sulfonato-a-L-idopyranosyl-
uronate)-(1?4)-(2-azido-3-O-benzvl-2-deoxv-6-Q-lithium sulfonato-a-D-
qlucopyranosylHd ->4)-(lithium 3-Q-benzvl-2-O-lithium sulfonato-a-L-
idopvranosyluronate)-(1-»4M2-azido-3-O-benzvl-2-deoxv-6-Q-lithium sulfonato-
g-D-qlucopyranosyiyb-d —>4)-(lithium 3-Q-benzyl-2-O-lithium sulfonato-g-L-
idopvranosyluronate)-(1?4)-3-O-benzvl-2-benzyloxvcarbonvlamino-2-deoxv-6-
O-lithium sulfonato-a-D-qlucopyranoside (110)
To a solution of compound 109 (57.3 mg, 13.7 umol) in a 1/1 v/v
tetrahydrofuran/methanol mixture (2.2 ml_) is added, at 0°C, a 0.7 M solution of
lithium hydroxide in water (0.88 ml_; qs final concentration of 0.2 M). After 1 hour
at 0°C and then 16 hours at room temperature, the reaction medium is deposited
on a column of Sephadex® LH20 (120 x 3 cm) eluted with a 50/50/1 v/v/v
dichloromethane/ethanol/water mixture to give compound 110 (38.1 mg).
[a]D 13.1° (c 1.0; MeOH)
Methyl (sodium 2-O-sodium sulfonato-a-L-idopvranosyluronate)-(1-»4H2-
amino-2-deoxy-6-Q-sodium sulfonato-a-D-glucopyranosyl)-f(1 -»4Msodium 2-O-
sodium sulfonato-a-L-idopyranosyluronateH1^4H2-amino-2-deoxy-6-Q-sodium
sulfonato-a-D-alucopyranosvOlp-d ->4)-(sodium 2-O-sodium sulfonato-g-L-
idopyranosyluronate)-(1?4)-2-amino-2-deoxy-6-Q-sodium sulfonato-a-D-
glucopyranoside (111)
To a solution of compound 110 (40 mg, 12.8 umol) in a 1/1 v/v tert-
butanol/water mixture (2.6 mL) are added ammonium formate (105 mg,
1.67 mmol) and 10% palladium-on-charcoal (260 mg). After stirring for 4 hours at
room temperature, the reaction medium is filtered (Millipore® LSWP 5 urn filter)
and concentrated to dryness. The residue is deposited on a column of
Sephadex® G25-fine gel (95 x 2 cm) eluted with aqueous 0.2 M NaCI solution.
The fractions containing the expected compound are combined and deposited
on a column of Sephadex® G25-fine gel (95 x 2 cm) eluted with water. The crude
product 111 thus obtained (14.7 mg) is used without further purification in the
following step.
Mass: "ESI" method, negative mode: theoretical mass = 2285.47;
experimental mass: 2197.20 ± 0.34 a.m.u. (iduronic acids observed in COOH
form).
Examples of compounds according to the invention:
EXAMPLE 1: Methyl (sodium 4-O-propvl-2-Q-sodium sulfonato-a-L-
idopvranosyluronateM1->4)-(2-deoxv-6-Q-sodium_________sulfonato-2-sodium
(sulfonatoamino)-a-D-qlucopvranosyl)-(1-*4H(sodium 2-O-sodium sulfonato-a-L-
idopvranosyluronate)-(1?4)-(2-deoxv-6-O-sodium_________sulfonato-2-sodium
(sulfonatoamino)-a-D-qlucopvranosyl-(l ->4 ^-(sodium 2-O-sodium sulfonato-a-L-
idopvranosyluronate)-(1?4)-2-deoxv-6-O-sodium__________sulfonato-2-sodium
(sulfonatoamino)-a-D-qlucopyranoside (compound 1)
To a freshly prepared solution of compound 30 (180 mg, 0.077 mmol) in
saturated aqueous sodium hydrogen carbonate solution (15 mL, 100 mL/mmol)
are added, at 0°C and under an argon atmosphere, solid sodium hydrogen
carbonate (1.17 g, 13.9 mmol) and then the pyridine-sulfur trioxide complex
(985 mg, 6.19 mmol) portionwise over 30 minutes. After 16 hours at room
temperature, the reaction mixture is deposited on a column of Sephadex® G25-
fine gel (90 x 3 cm) eluted with aqueous 0.2 M NaCI solution. The fractions
containing the expected compound are combined and deposited on a column of
Sephadex® G25-fine gel (90 x 3 cm) eluted with water. After concentrating the
fractions containing the expected compound, 200 mg of compound 1 are
obtained.
1H NMR [600 MHz] (D20) 5 of the anomeric protons: 5.45; 5.42 (2 H);
5.22; 5.21; 5.20; 5.18; 5.03 ppm.
Mass: "ESI" method, negative mode: theoretical mass = 2735.72;
experimental mass: 2734 a.m.u.
EXAMPLE 2: Methyl (sodium 4-Q-propvl-2-Q-sodium sulfonato-a-L-
idopyranosyluronate)-(1?4)-(2-deoxv-6-O-sodium_________sulfonato-2-sodium
(sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-r(sodium 2-O-sodium sulfonato-a-L-
idopvranosyluronate)-(1?4)-(2-deoxv-6-O-sodium_________sulfonato-2-sodium
(sulfonatoamino)-a-D-qlucopvranosyl-(l->4)1:r(sodium 2-O-sodium sulfonato-a-L-
idopvranosyluronate)-(1?4)-2-deoxy-6-Q-sodium__________sulfonato-2-sodium
(sulfonatoamino)-a-D-qlucopyranoside (compound 2)
Compound 37 (29.5 mg, 11.56 umol) is treated according to the same
procedure as that described for the preparation of Example 1 to give compound
2 (7.4 mg) after purification by ion-exchange chromatography (SAX column,
conditions: 0.5 mL/min, A: H20, B: 2 M NaCI, gradient 30% of B to 90% over
30 min) followed by chromatography on a column of Sephadex® G25-fine gel (54
x 1.7 cm, water).
1H NMR [600 MHz] (D20) 5 of the anomeric protons: 5.45; 5.42; 5.41
(2 H); 5.22; 5.21 (2H); 5.20; 5.18; 5.03 ppm.
Mass: "ESI" method, negative mode: theoretical mass = 3401.12;
experimental mass: 3400.40 ± 0.76 a.m.u.
EXAMPLE 3: Sodium rmethyl (sodium 2-O-sodium sulfonato-a-L-
idopvranosyluronateH1->4)-(2-deoxv-6-O-sodium_________sulfonato-2-sodium
(sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-F(sodium 2-O-sodium sulfonato-a-L-
idopyranosyluronate)-(1?4)-(2-deoxy-6-O-sodium_________sulfonato-2-sodium
(sulfonatoamino)-a-D-glucopyranosyl)-(1?4)1?-2-Q-sodium_______sulfonato-a-L-
idopyranosideluronate (compound 3)

Crude compound 55 (48.7 mg) is treated according to the same
procedure as that described for the preparation of Example 1 to give
compound 3 (37.6 mg).
1H NMR [600 MHz] (D20) 6 of the anomeric protons: 5.43 (2H); 5.35;
5.25; 5.24; 5.19; 5.18; 5.09 ppm.
EXAMPLE 4: Methyl (sodium 2-Q-sodium sulfonato-a-L-
idopyranosyluronate)-(1?4)-(2-deoxy-2-sodium__________(sulfonatoamino)-a-D-
qlucopvranosyl)-(1?4)-(sodium 2-O-sodium sulfonato-g-L-idopyranosyluronate)-
(1?4)-(2-deoxv-2-sodium (sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium
2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-deoxv-6-Q-sodium
sulfonato-2-sodium (sulfonatoamino)-a-D-qlucopvranosyl)-(1?4)-(sodium 2-O-
sodium_________sulfonato-a-L-idopvranosyluronate)-(1?4)-2-deoxv-6-Q-sodium
sulfonato-2-sodium (sulfonatoamino)-a-p-glucopyranoside (compound 4)

Compound 91 (5.5 mg, 2.64 umol) is treated according to the same
procedure as that described for the preparation of Example 1 to give compound
4 (4.5 mg).
1H NMR [600 MHz] (D20) 5 of the anomeric protons: 5.44; 5.40; 5.31;
5.25; 5.24; 5.22; 5.16; 5.03 ppm.
Mass: "ESI" method, negative mode: theoretical mass = 2489.55;
experimental mass: 2465.63 ± 0.64 a.m.u.
EXAMPLE 5: Methyl (sodium 2-O-sodium sulfonato-a-L-
idopvranosyluronate)-(1?4)-(2-deoxv-2-sodium__________(sulfonatoamino)-a-D-
glucopvranosyl)-(1?4)-(sodium 2-O-sodium sulfonato-g-L-idopyranosyluronate)-
(1?4)-(2-deoxv-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-g-D-
glucopyranosvP-d ->4)-(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-
(1 ->4M2-deoxv-2-sodium (sulfonatoamino)-g-D-glucopvranosyl)-(1?4)-(sodium
2-O-sodium sulfonato-g-L-idopyranosyluronate)-(1?4)-2-deoxv-6-Q-sodium
sulfonato-2-sodium (sulfonatoamino)-a-D-glucopyranoside (compound 5)

Compound 95 (2.5 mg, 1.2 umol) is treated according to the same
procedure as that described for the preparation of Example 1 to give compound
5(1.9mg).
1H NMR [600 MHz] (D20) 8 of the anomeric protons: 5.45; 5.43; 5.33;
5.25; 5.23; 5.21; 5.16; 5.05 ppm.
EXAMPLE 6: Methyl (sodium 2-O-sodium sulfonato-a-L-
idopyranosyluronate)-(1-»4H2-deoxv-6-O-sodium_________sulfonato-2-sodium
(sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-sodium sulfonato-a-L-
idopvranosyluronate)-(1?4)-(2-deoxv-2-sodium__________(sulfonatoamino)-a-D-
glucopyranosylMI ->4Msodium 2-O-sodium sulfonato-g-L-idopyranosyluronate)-
(1?4)-(2-deoxv-2-sodium (sulfonatoamino)-g-D-glucopvranosyl)-(1?4)-(sodium
2-O-sodium sulfonato-a-L-idopvranosyluronate)-(1?4)-2-deoxv-6-O-sodium
sulfonato-2-sodium (sulfonatoamino)-a-D-glucopyranoside (compound 6)

Compound 99 (8.9 mg, 4.28 umol) is treated according to the same
procedure as that described for the preparation of Example 1 to give compound
6 (7.3 mg).
1H NMR [600 MHz] (D20) 8 of the anomeric protons: 5.46; 5.32 (2 H);
5.26; 5.25; 5.21; 5.20; 5.05 ppm.
Mass: "ESI" method, negative mode: theoretical mass = 2489.55;
experimental mass: 2488.00 ±1.5 a.m.u.
EXAMPLE 7: Methyl (sodium 2-O-sodium sulfonato-g-L-
idopvranosyluronate)-(1?4)-(2-deoxy-6-Q-sodium_________sulfonato-2-sodium
(sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-sodium sulfonato-g-L-
idopvranosyluronateM1->4)-(2-deoxy-2-sodium_________(sulfonatoamino)-g-D-
glucopyranosylMI ->4Hsodium 2-O-sodium sulfonato-g-L-idopyranosyluronate)-
(1?4)-(2-deoxv-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-g-D-
glucopyranosyl)-(1?4)-(sodium 2-Q-sodium sulfonato-g-L-idopyranosyluronate)-
(1?4)-2-deoxy-6-O-sodium_____sulfonato-2-sodium_____(sulfonatoamino)-g-p-
glucopyranoside (compound 7)

Compound 108 (3.7 mg, 1.42 umol) is treated according to the same
procedure as that described for the preparation of Example 1 to give compound
7(1.8mg).
1H NMR [600 MHz] (D20) 8 of the anomeric protons: 5.46; 5.42; 5.31;
5.26; 5.24; 5.22; 5.18; 5.03 ppm.
EXAMPLE 8: Methyl (sodium 2-O-sodium sulfonato-a-L-
idopvranosyluronate)-(1?4)-(2-deoxv-6-O-sodium_________sulfonato-2-sodium
(sulfonatoamino)-g-D-qlucopyranosyl)-f(1?4)-(sodium 2-O-sodium sulfonato-a-L-
idopvranosyluronate)-(1?4)-(2-deoxv-6-O-sodium_________sulfonato-2-sodium
(sulfonatoaminoV-g-D-glucopyranosyiyb-d ->4)-(sodium 2-O-sodium sulfonato-a-
L-idopvranosyluronate)-(1?4)-2-deoxv-6-O-sodium______________sulfonato-2-
(sulfonato)amino-a-D-qlucopyranoside (compound 8)

Compound 111 (42 mg, 18.4 umol) js treated according to the same
procedure as that described for the preparation of Example 1 to give compound
8(44.1 mg).
1H NMR [600 MHz] (D20) 5 of the anomeric protons: 5.45; 5.44; 5.25;
5.24 (2H); 5.20; 5.04 ppm.
Mass: "ESI" method, negative mode: theoretical mass = 2693.84;
experimental mass: 2692.97 ±0.18 a.m.u.
EXAMPLE 9: Methyl (sodium 2-O-sodium sulfonato-g-L-
idopvranosyluronate)-(1?4)-(2-deoxv-6-O-sodium_________sulfonato-2-sodium
(sulfonato)amino-g-D-glucopyranosyl)-r(1?4)-(sodium 2-O-sodium sulfonato-g-L-
idopvranosyluronate)-(1?4)-(2-deoxv-6-Q-sodium_________sulfonato-2-sodium
(sulfonatoamino)-g-D-qlucopvranosyl)1?-(1?4)-(sodium 2-O-sodium sulfonato-g-
L-idopvranosyluronate)-(1?4)-2-deoxv-2-sodium_________(sulfonatoamino)-g-P-
qlucopyranoside (compound 9)

Compound 104 (6.5 mg, 2.98 umol) is treated according to the same
procedure as that described for the preparation of Example 1 to give compound
9 (6.0 mg).
1H NMR [600 MHz] (D20) 8 of the anomeric protons: 5.44 (2H); 5.43; 5.22
(2H); 5.19; 5.18; 5.05 ppm.
Mass: "ESI" method, negative mode: theoretical mass = 2591.60;
experimental mass: 2591.80 ± 0.33 a.m.u.
The compounds according to the invention underwent pharmacological
trials to determine their agonist effect on the FGF receptors and their activity on
angiogenesis and also on post-ischaemic revascularization.
Model of in vitro anqioqenesis: specific activity towards FGF2
The in vitro angiogenesis model corresponds to a rearrangement of
human venous endothelial cells on a biological matrix. The matrix is made by
dispensing, into each well of a 96-well plate (Becton Dickinson 353872), 60 pi of
Matrigel® diluted to 1/3 (Growth factor reduced Matrigel®: Becton Dickinson
356230) in collagen (rat tail collagen, type I: Becton Dickinson 354249). The
biological matrix hardens after 1 hour at 37°C.
Human venous endothelial cells (HUVEC ref: C-12200 - Promocell) are
seeded onto the biological matrix at 7800 cells/well in 120 pi of EBM® medium
(Endothelial Basal Medium, Lonza C3121) + 2% FCS (foetal calf serum - Lonza)
+ hEGF (Recombinant Human Epidermal Growth Factor - Lonza) 10 pg/ml. The
cells are stimulated with FGF2 (R&D Systems 7 234 - FSE - 0 50) 10 ng/ml or
with the products of the invention for 18 hours at 37°C in the presence of 5%
CO2. After 24 hours, the cells are observed under a microscope (X4 objective
lens) and analysis of the length of the pseudo-tubules is performed with the aid
of image software (Biocom VisioLab 2000 software).
In this test of in vitro angiogenesis, the compounds of the invention have a
specific activity of between 10"6 M and 10"12 M. For example, compounds 1 and 7
are active at 10"6M.
tested in sodium salt form, also demonstrated activity in this in vitro
angiogenesis test.
Moreover, it was demonstrated in an in vitro cellular test that the
octasaccharide No. 8 according to the invention, the heptasaccharide No. 3 and
the decasaccharide No. 2 are better FGF-2 activators than the hexasaccharide
analogue thereof (compound described by C. Tabeur et al. in Bioorg. & Med.
Chem., 1999, 7, 2003-2012). Furthermore, the majority of the other
octasaccharide compounds according to the invention, for instance compound
No. 7, have the same activity as the octasaccharide No. 8 on in vitro models.
Model of cellulose implant in mice
This model is an adaptation of the model described by Andrade et al.
(Microvascular Research, 1997, 54, 253-61) for testing pharmacological
products capable of activating the onset of angiogenesis.
The animals (white consanguineous BALB/c J mice) are anaesthetized
with a xylazine (Rompun®, 10 mg/kg)/ketamine (Imalgene® 1000, 100 mg/kg)
mixture intraperitoneal^. The animal's back is shaved and disinfected with
Hexomedine®. An air pocket is created subcutaneously on the mouse's back by
injecting 5 ml of sterile air. An incision of about 2 cm at the top of the animal's
back is made in order to introduce a sterile cellulose implant (disk 1 cm in
diameter, 2 mm thick, Cellspon® ref. 0501) impregnated with 50 ul of sterile
solution containing the test product. The incision is then sutured and cleaned
with Hexomedine®.
On the days following the insertion of the implant, the mice can receive
the product into the implant via an injection through the skin (50 ul/implant/day)
under gaseous anaesthesia (5% isoflurane (Aerrane®, Baxter)).
Seven days after inserting the sponge, the mice are sacrificed by means
of a lethal dose of pentobarbital sodium (CEVA Sante Animale), administered
intraperitoneally. The skin is then excised, about 1 cm around the sponge, while
avoiding the scar, so as to release the skin and the sponge. The sponge is then
cut into several pieces and placed in a Ribolyser® containing 1 ml of lysis buffer
(Cell Death Detection ELISA, Roche). The tubes are shaken four times
consecutively, for 20 seconds, at force 4, using a cell mill (FastPrep® FP 120).
The tubes are then centrifuged for 10 minutes at 2000xg at 20°C and the
supernatants are frozen at -20°C until the time of the haemoglobin assay. On the
day of the assay, the tubes are again centrifuged after thawing and the
haemoglobin concentration is measured with the Drabkin reagent (Sigma,
volume for volume) by reading on a spectrophotometer at 405 nm against a
standard range of bovine haemoglobin (Sigma).
The haemoglobin concentration in each sample is expressed in mg/ml
from the polynomial regression produced from the range. The results are
expressed as a mean value (± sem) for each group. The differences between
the groups are tested with an ANOVA followed by a Dunnett test on the square
root of the values.
In this in vivo test, the compounds of the invention revealed a specific
activity of between 5 and 45 ng/site. Thus, compounds 1 and 7 are active at a
concentration of 45 ng/site.
It therefore appears that the compounds according to the invention have
agonist activity on the FGF receptors and activity on angiogenesis, and also on
post-ischaemic revascularization. These compounds may thus be used for the
preparation of medicaments, especially medicaments that are useful for treating
diseases that require activation of the FGF receptors, or of medicaments that are
useful in pathologies requiring activation of angiogenesis post-ischaemic
revascularization.
Thus, according to another of its aspects, a subject of the invention is
thus medicaments that comprise a compound of formula (l)/(l') or compound 10,
or a pharmaceutically acceptable salt thereof.
One subject of the invention is, more generally, medicaments comprising
a compound of formula (I):

in which:
- Ri represents a group -OSO3" or a hydroxyl group,
- R2 represents either a group -O-alkyl, or a monosaccharide of formula
(II), in which R represents an alkyl group:

- R3 represents a disaccharide of formula (III):

in which:
- R4 represents a group -OSO3- or a hydroxyl group,
- R5 represents a disaccharide of formula (IV):

in which:
- R6 represents a group -OSO3- or a hydroxyl group,
- R7 represents either a hydroxyl group or a monosaccharide of
formula (V) below, or a disaccharide of formula (VI):

in which:
- R8 represents a group -OSO3- or a hydroxyl group,
- R9 represents either a hydroxyl group or a group -O-alkyl, or a
disaccharide of formula (VII):

in which R10 represents a group -O-alkyl,
on condition that: R9 represents a hydroxyl group or a group -O-alkyl when
R2 represents a monosaccharide of formula (II) as defined above; R7 represents
a disaccharide of formula (VI) as defined above when R2 represents a group
-O-alkyl; and R1, R4, R6 and R8 do not simultaneously represent hydroxyl groups.
Such compounds include those of formula (l)/(l') defined above, and also
the heptasaccharide 10 defined previously, described in patent application
US 2006/0079483 A1.
These medicaments find their use in therapy, especially in the treatment
of ischaemia (cardiac ischaemia, arterial ischaemia of the lower limbs), the
treatment of diseases associated with narrowing or obstruction of the arteries or
arterites, the treatment of angina pectoris, the treatment of thromboangitis
obliterans, the treatment of atherosclerosis, the treatment of inhibition of
restenosis after angioplasty or endoarterectomy, the treatment of cicatrization,
muscle regeneration treatment, treatment for the survival of myoblasts, the
treatment of peripheral neuropathy, the treatment of post-operative nerve
damage, the treatment of nerve deficiencies such as Parkinson's disease,
Alzheimer's disease, prion disease and neuronal degeneration in alcoholics, the
treatment of dementias, treatment for improving the survival of a bioartificial
pancreas graft in the case of diabetics, treatment for improving the
revascularization of grafts and the survival of grafts, the treatment of retinal
degeneration, the treatment of pigmentary retinitis, the treatment of
osteoarthritis, the treatment of pre-eclampsia or the treatment of vascular lesions
and of acute respiratory distress syndrome, treatment for cartilage repairing,
treatment for repairing and protecting bones, treatment for repairing and
protecting hair follicles and for protecting and regulating hair growth.
Ischaemia is a decrease in arterial circulation in an organ, leading to a
decrease in oxygen concentration in the damaged tissues. In the mechanisms of
post-ischaemic revascularization, two main mechanisms are involved:
angiogenesis and arteriogenesis. Angiogenesis is the process of generating new
blood capillaries from pre-existing vessels. Arteriogenesis contributes towards
the development (increase in size and calibre) of the collateral vessels around
the ischaemic or avascular area.
Among the growth factors involved in these revascularization processes,
the FGF family and especially FGF-2 has been the most widely described (Post,
M. J., Laham, R., Sellke, F. W. & Simons, M. Therapeutic angiogenesis in
cardiology using protein formulations. Cardiovasc. Res. 49, 522-31, 2001).
Thus, FGF2 and its receptors represent very pertinent targets for
therapies directed towards inducing angiogenesis and arteriogenesis processes
(Khurana, R. & Simons, M. Insights from angiogenesis trials using fibroblast
growth factor for advanced arteriosclerotic disease. Trends Cardiovasc. Med. 13,
116-22,2003).
One of the applications of the compounds of the invention is post-
ischaemic treatment after heart occlusion or occlusion of the peripheral arteries.
As regards the treatment of cardiac ischaemia, one of the most promising clinical
tests is a clinical test in which FGF-2 was sequestered in alginate microspheres
in the presence of heparin (Laham, R. J. et al. Local perivascular delivery of
basic fibroblast growth factor in patients undergoing coronary bypass surgery:
results of a phase I randomized, double-blind, placebo-controlled trial.
Circulation 100, 1865-71, 1999). These microspheres were implanted close to
the ischaemic locus in the myocardium. After 90 days, all the patients treated
with FGF2 showed no ischaemic cardiac symptoms. In comparison, in the
control group, three of the seven patients had persistent symptoms at 90 days
and two patients required vascular surgery. Interestingly, the therapeutic benefit
was maintained after 3 years of monitoring. These observations suggest that
compounds that mimic FGF2 may represent a therapy of choice for treating the
consequences of cardiac ischaemia.
Three clinical tests on the injection of FGF2 into the coronary artery were
performed during treatment of narrowing of the coronary arteries (Laham, R. J.
et al. Intracoronary basic fibroblast growth factor (FGF-2) in patients with severe
ischemic heart disease: results of a phase I open-label dose escalation study. J.
Am. Coll. Cardiol. 36, 2132-9, 2000; Simons, M. et al. Pharmacological
treatment of coronary artery disease with recombinant fibroblast growth factor-2:
double-blind, randomized, controlled clinical trial. Circulation 105, 788-93, 2002;
Unger, E. F. et al. Effects of a single intracoronary injection of basic fibroblast
growth factor in stable angina pectoris. Am. J. Cardiol. 85, 1414-9, 2000). The
result of these three tests shows that intra-coronary infusions of FGF2 are well
tolerated and significantly improve the condition of the patients. Thus, the
compounds described in the invention may find an application in the treatment of
diseases associated with narrowing of the coronary arteries and especially in the
treatment of angina pectoris.
Diseases of the distal arteries and especially arteritis of the lower limbs
are caused by chronic obstruction of the arterioles that irrigate the extremities.
These pathologies mainly affect the lower limbs. In a phase I clinical trial,
patients with peripheral artery pathologies leading to claudication received
injections of FGF2 (Lazarous, D. F. et al., Basic fibroblast growth factor in
patients with intermittent claudication: results of a phase I trial. J. Am. Coll.
Cardiol. 36, 1239-44, 2000). In this context, FGF2 was well tolerated in these
patients and the clinical data suggest a beneficial effect of FGF2 and especially
on improving walking. These clinical data suggest that the compounds of the
invention represent a therapeutic tool of choice for the treatment of diseases
associated with obstruction of the distal arteries.
Buerger's disease or thromboangitis obliterans affects the distal vascular
structures and is characterized by distal arteritis of the legs, with pain and
ulceration. In this context, an induction of angiogenesis and of vasculogenesis
would represent a therapy for this pathology. The compounds of the said
invention represent a therapy of choice for thromboangitis obliterans.
Peripheral neuropathy is an axonal or demyelinizing attack of the motor
and/or sensory peripheral nerve which leads to desensitization of the distal
limbs. One of the major secondary complications of diabetes is the chronic
development of peripheral neuropathy. In this context, it has been demonstrated
that FGF2 induces axonal regeneration, which might be a therapy of choice in
the treatment of peripheral nerve lesion and thus in peripheral neuropathy (Basic
fibroblast growth factor isoforms promote axonal elongation and branching of
adult sensory neurons in vitro. Klimaschewski L, Nindl W, Feurle J, Kavakebi P,
Kostron H. Neuroscience. 2004;126(2):347-53). By virtue of the agonist activity
on the FGF receptors, the compounds of the said invention would represent a
treatment of choice in peripheral neuropathy in the case of healthy or diabetic
patients.
It is clearly established that FGF2 is an activator of nerve cells during
development. Recent results suggest that FGF2 is also a pivotal factor for
promoting the regeneration of neurons in adults (Sapieha PS, Peltier M, Rendahl
KG, Manning WC, Di Polo A., Fibroblast growth factor-2 gene delivery stimulates
axon growth by adult retinal ganglion cells after acute optic nerve injury. Mol.
Cell. Neurosci. 2003 Nov.; 24(3):656-72.). By virtue of their agonist activities on
the FGF receptors, the compounds of the said invention would represent a
treatment of choice in repairing post-operative nerve damage, in repairing nerve
deficiencies such as Parkinson's disease, Alzheimer's disease, prion disease
and neuronal degeneration in alcoholics or in the case of dementia.
The proliferation and migration of vascular smooth muscle cells
contributes towards intimal hypertrophy of the arteries and thus plays a
predominant role in atherosclerosis and in restenosis after angioplasty and
endoarterectomy. It has been demonstrated that an angiogenic factor, VEGF,
significantly reduces the thickening of the intima by accelerating re-
endothelialization (Van Belle, E., Maillard, L, Tio, F. O. & Isner, J. M.
Accelerated endothelialization by local delivery of recombinant human vascular
endothelial growth factor reduces in-stent intimal formation. Biochem. Biophys.
Res. Commun. 235, 311-6, 1997). Thus, the compounds of the present
invention, with pro-angiogenic activity, may be useful in treatment of
atherosclerosis and in inhibiting restenosis after angioplasty or endoarterectomy.
The vascular network is essential to the development and maintenance of
tissues. By promoting the delivery of nutrients, oxygen and cells, the blood
vessels help to maintain the functional and structural integrity of tissues. In this
context, angiogenesis and vasculogenesis make it possible to preserve and to
perfuse tissues after ischaemia. The angiogenic growth factors such as VEGF
and FGF2 thus promote revascularization for tissue regeneration. The
compounds presented in the invention could represent a treatment of choice in
muscle regeneration treatment.
The processes of muscle regeneration on dystrophic or normal muscles
depend on the supply of cytokines and of angiogenic growth factors at the local
level (Fibbi, G., D'Alessio, S., Pucci, M., Cerletti, M. & Del Rosso, M. Growth
factor-dependent proliferation and invasion of muscle satellite cells require the
cell-associated fibrinolytic system. Biol. Chem. 383, 127-36, 2002). It has been
proposed that the FGF system is a critical system of muscle regeneration and of
myoblast survival and proliferation (Neuhaus, P. et al. Reduced mobility of
fibroblast growth factor (FGF)-deficient myoblasts might contribute to dystrophic
changes in the musculature of FGF2/FGF6/mdx triple-mutant mice. Mol. Cell.
Biol. 23, 6037-48, 2003). FGF2 and the compounds of the said invention could
be exploited in order to promote cardiac regeneration. They would thus improve
the perfusion of the myocardium after ischaemia (Hendel, R. C. et al. Effect of
intracoronary recombinant human vascular endothelial growth factor on
myocardial perfusion: evidence for a dose-dependent effect. Circulation 101,
118-21, 2000) and also the survival and progress of transplanted myoblasts,
especially in Duchenne's muscular dystrophy.
Angiogenesis is an essential phenomenon during cutaneous cicatrization.
The new vessels formed supply the oxygen and nutrients required for tissue
repair. In the case of diabetics, cicatrization is a slow and difficult process
presenting angiogenesis defects. FGFs are among the growth factors that are
the most involved in angiogenesis processes during the cicatrization phase.
Certain FGFs are highly overregulated in dermal cells after a cutaneous injury.
On account of their agonist activity on the FGF receptors, the compounds of the
said invention would represent a therapy of choice for the treatment of
cicatrization in healthy or diabetic patients.
Bioartificial pancreas transplantation is a very promising technique for the
treatment of certain types of diabetes. It has been demonstrated, in diabetic rats,
that vascularisation in bioartificial pancreases is much greater when the
pancreases are impregnated with microspheres bearing FGF2 (Sakurai,
Tomonori; Satake, Akira, Sumi, Shoichiro, Inoue, Kazutomo, Nagata, Natsuki,
Tabata, Yasuhiko. The Efficient Prevascularization Induced by Fibroblast Growth
Factor 2 With a Collagen-Coated Device Improves the Cell Survival of a
Bioartificial Pancreas. Pancreas. 28(3):e70-e79, April 2004). This
revascularization thus improves the survival of implanted bioartificial pancreases
and consequently the survival of the graft. By virtue of their agonist activities on
the FGF receptors, the compounds of the said invention would represent a
therapy of choice in improving the survival of bioartificial pancreas grafts in
diabetics and more generally in improving the revascularization of grafts and
consequently the survival of the grafts.
Pigmentary retinitis is a pathology involving progressive degeneration of
the retina characterized by degeneration of the photoreceptors and obliteration
of the retinal vessels. Lahdenranta et al. (An anti-angiogenic state in mice and
humans with retinal photoreceptor cell degeneration. Proc. Natl. Acad. Sci. USA
98, 10368-73, 2001) have proposed that angiogenic growth factors regulate the
neural coordination and the associated vascularization of the retina by
simultaneously functioning as photoreceptor survival factors and as endothelial
cell regulators. In this context, the intra-vitreal injection of FGF2 retards the
degeneration of the photoreceptors by acting on retinal survival and retinal
angiogenesis (Faktorovich, E. G., Steinberg, R. H., Yasumura, D., Matthes, M. T.
& LaVail, M. M. Basic fibroblast growth factor and local injury protect
photoreceptors from light damage in the rat. J. Neurosci. 12, 3554-67,1992).
These observations demonstrate the interest of the compounds described in the
invention as a therapy in retinal degeneration and especially in pigmentary
retinitis.
In the field of osteoarthritis, many studies have been performed for
restoring destroyed articular cartilage. In this context, it has been reported that
the proliferation and differentiation of chondrocytes were stimulated by FGF2 in
vitro (Kato Y, Gospodarowicz D. Sulfated proteoglycan synthesis by confluent
cultures of rabbit costal chondrocytes grown in the presence of fibroblast growth
factor. J. Cell Biol. 1985 Feb.; 100(2):477-85). Furthermore, Cuevas et al. have
shown that FGF2 induces cartilage repair in vivo (Cuevas P, Burgos J, Baird A.
Basic fibroblast growth factor (FGF) promotes cartilage repair in vivo. Biochem.
Biophys. Res. Commun. 1988 Oct. 31;156(2):611-8). Takafuji et al. have also
shown that FGF2 implants significantly improve temporo-mandibular cartilage in
rabbits suffering from osteoarthritis (Takafuji H, Suzuki T, Okubo Y, Fujimura K,
Bessho K Regeneration of articular cartilage defects in the temporomandibular
joint of rabbits by fibroblast growth factor-2: a pilot study. Int. J. Oral Maxillofac.
Surg. 2007 Oct.; 36(10):934-7). These observations demonstrate the interest the
compounds described in the invention as a therapy in treatment of osteoarthritis
and cartilage repair.
In the field of bone repair, one of the essential needs is to find agents that
stimulate bone formation. Among the main growth factors, it is established that
the systemic administration of FGF2 facilitates bone repair (Acceleration of
fracture healing in nonhuman primates by fibroblast growth factor-2. Kawaguchi
H, Nakamura K, Tabata Y, Ikada Y, Aoyama I, Anzai J, Nakamura T, Hiyama Y,
Tamura M. J. Clin. Endocrinol. Metab. 2001 Feb.; 86(2), 875-880). The local
application of FGF2 in gelatin matrices accelerates bone repair in primates,
suggesting the clinical utility of FGF2 in the treatment of fractures. By virtue of
their agonist properties for the FGF receptors, the compounds of the said
invention would represent a treatment of choice in bone repair.
Pre-eclampsia is a pathology of the placenta associated with a
vascularization defect (Sherer, D. M. & Abulafia, O. Angiogenesis during
implantation, and placental and early embryonic development. Placenta 22, 1-
13, 2001). These vascularization defects are thought to be due to an
angiogenesis defect and lead to placental disruptions that may result in the
death of the foetus. The compounds of the invention may be a treatment of
choice for overcoming an angiogenesis defect in pre-eclamptic placentas.
In addition to angiogenesis-inducing effects, growth factors such as VEGF
or FGF2 protect endothelial cells against intrinsic and extrinsic apoptosis
inducers. The intrinsic signalling pathway is activated by the mitochondria in
response to a stress such as deprivation or DNA damage, whereas the extrinsic
signalling pathway is induced by the binding of pro-apoptotic factors such as
TNF-a or Fas. It is now clearly described that VEGF and FGF2 are two factors of
endothelial cell survival (Role of Raf in Vascular Protection from Distinct
Apoptotic Stimuli: A Alavi, J.D. Hood, R. Frausto, D. G. Stupack, D.A. Cheresh:
Science 4 July 2003: Vol. 301. No. 5629, pp. 94-96). Acute respiratory distress
syndrome (ARDS) is characterized by cardiovascular and neuropsychiatric
problems, in the context of the cardiovascular problems, the patients present
major vascular lesions and especially a high induction of apoptosis of
endothelial cells. Recently, Hamacher et al. have demonstrated that the
bronchoalveolar lavage fluids of patients suffering from ARDS showed pro-
apoptotic activity against lung microvascular endothelial cells (Tumor necrosis
factor-alpha and angiostatin are mediators of endothelial cytotoxicity in
bronchoalveolar lavages of patients with acute respiratory distress syndrome.
Am. J. Respir. Crit. Care Med. 2002 Sep. 1;166(5):651-6: Hamacher J., Lucas
R., Lijnen H.R., Buschke S., Dunant Y., Wendel A., Grau G.E., Suter P.M.,
Ricou B.). By virtue of their activity on endothelial cell survival, the products of
the invention might be a treatment of choice in the vascular improvement of
patients suffering from vascular lesions and especially patients suffering from
ARDS.
The endogenous overregulation of FGF7 (or KGF) and of FGF18 appears
to be an important mechanism for promoting the proliferation, migration and
protection of hair follicles in pathological cases or after a tumoral treatment
(Comprehensive Analysis of FGF and FGFR Expression in Skin: FGF18 Is
Highly Expressed in Hair Follicles and Capable of Inducing Anagen from
Telogen Stage Hair Follicles. Mitsuko Kawano, Akiko Komi-Kuramochi,
Masahiro Asada, Masashi Suzuki, Junko Oki, Ju Jiang and Toru Imamura). By
virtue of their agonist activity on the FGF receptors, the compounds of the said
invention might be a treatment of choice for repairing and protecting hair follicles
and for protecting and regulating hair growth.
According to another of its aspects, the present invention relates to
pharmaceutical compositions comprising, as active principle, a compound
according to the invention or a compound 10. These pharmaceutical
compositions contain an effective dose of at least one compound according to
the invention or of compound 10, or a pharmaceutically acceptable salt of the
said compound, and also at least one pharmaceutically acceptable excipient.
The said excipients are chosen, according to the pharmaceutical form and the
desired mode of administration, from the usual excipients known to those skilled
in the art.
Thus, one subject of the invention is a pharmaceutical composition
comprising, as active principle, at least one compound of formula (I) in which:
- R1 represents a group -OSO3" or a hydroxyl group,
- R2 represents either a group -O-alkyl, or a monosaccharide of formula
(II), in which R represents an alkyl group:
- R3 represents a disaccharide of formula (III):
in which:
- R4 represents a group -OSO3- or a hydroxyl group,
- R5 represents a disaccharide of formula (IV):

in which:
- R6 represents a group -OSO3" or a hydroxyl group,
- R7 represents either a hydroxyl group or a monosaccharide of
formula (V) below, or a disaccharide of formula (VI):

in which:
- R8 represents a group -OSO3" or a hydroxyl group,
- R9 represents either a hydroxyl group or a group -O-alkyl, or a
disaccharide of formula (VII):

in which R10 represents a group -O-alkyl,
on condition that: R9 represents a hydroxyl group or a group -O-alkyl when
R2 represents a monosaccharide of formula (II) as defined above; R7 represents
a disaccharide of formula (VI) as defined above when R2 represents a group
-O-alkyl; and R1, R4, R6 and R8 do not simultaneously represent hydroxyl groups,
or a pharmaceutically acceptable salt of the said compound, and also at
least one pharmaceutically acceptable excipient.
Compounds of this kind encompass those of formula (l)/(l') defined
above, as well as heptasaccharide 10 defined above, which has been described
in patent application US 2006/0079483 A1.
In the pharmaceutical compositions of the present invention for oral,
sublingual, subcutaneous, intramuscular, intravenous, topical, local,
intratracheal, intranasal, transdermal or rectal administration, the active principle
above or the salt thereof may be administered in a unit administration form, as a
mixture with standard pharmaceutical excipients, to man and animals for the
prevention or treatment of the above disorders or diseases.
The appropriate unit administration forms include oral forms such as
tablets, soft or hard gel capsules, powders, granules and oral solutions or
suspensions, sublingual, buccal, intratracheal, intraocular, intranasal or
inhalation administration forms, topical, transdermal, subcutaneous,
intramuscular or intravenous administration forms, rectal administration forms
and implants. For topical application, the compounds according to the invention
may be used in creams, gels, ointments or lotions.
The injectable administration forms are particularly advantageous,
conventionally comprising the active compound dissolved in water for injection,
in the presence of sodium chloride. The unit dose of active compound should be
suited to the desired therapeutic effect; it may be, for example, between 0.1 and
100 mg of active principle.
According to another of its aspects, the present invention also relates to
the use of a compound according to the invention or of a compound 10, or a
pharmaceutically acceptable salt thereof, for treating the pathologies indicated
above.
Thus, one subject of the invention is a compound of formula (I) in which:
- R1 represents a group -OSO3- or a hydroxyl group,
- R2 represents either a group -O-alkyl, or a monosaccharide of
formula (II), in which R represents an alkyl group:

- R3 represents a disaccharide of formula (III):

in which:
- R4 represents a group -OSO3- or a hydroxyl group, and
- R5 represents a disaccharide of formula (IV):

in which:
- R6 represents a group -OSO3- or a hydroxyl group,
- R7 represents either a hydroxyl group or a monosaccharide of
formula (V) below, or a disaccharide of formula (VI):

in which:
- R8 represents a group -OSO3- or a hydroxyl group,
- R9 represents either a hydroxyl group or a group -O-alkyl, or a
disaccharide of formula (VII):

in which R10 represents a group -O-alkyl,
on condition that: R9 represents a hydroxyl group or a group -O-alkyl when
R2 represents a monosaccharide of formula (II) as defined above; R7 represents
a disaccharide of formula (VI) as defined above when R2 represents a group
-O-alkyl; and R1, R4, R6 and R8 do not simultaneously represent hydroxyl groups,
or a pharmaceutically acceptable salt of the said compound,
for treating the pathologies indicated above.
Compounds of this kind encompass those of formula (l)/(l') defined
above, as well as heptasaccharide 10 defined above, which has been described
in patent application US 2006/0079483 A1.
According to another of its aspects, the present invention also relates to a
method for treating the pathologies indicated above, which comprises the
administration to patients of an effective dose of a compound according to the
invention or of a compound 10 or a pharmaceutically acceptable salt thereof.
The medicaments, pharmaceutical compositions and treatment method
according to the invention may also concern any of the subgroups of compounds
defined previously.
CLAIMS
1. Oligosaccharide compounds corresponding to formula (I):

in which:
- R1 represents a group -OSO3- or a hydroxyl group,
- R2 represents either a group -O-alkyl, or a monosaccharide of
formula (II), in which R represents an alkyl group:

- R3 represents a disaccharide of formula (III):

in which:
- R4 represents a group -OSO3- or a hydroxyl group,
- R5 represents a disaccharide of formula (IV):

in which:
- R6 represents a group -OSO3" or a hydroxyl group,
- R7 represents either a hydroxyl group or a disaccharide of formula (VI):

in which:
- R8 represents a group -OSO3- or a hydroxyl group,
- Rg represents either a hydroxyl group or a group -O-alkyl, or a
disaccharide of formula (VII):

in which R10 represents a group -O-alkyl,
on condition that: R9 represents a hydroxyl group or a group -O-alkyl when
R2 represents a monosaccharide of formula (II) as defined above; R7 represents
a disaccharide of formula (VI) as defined above when R2 represents a group
-O-alkyl; and R1,R4, R6 and R8 do not simultaneously represent hydroxyl groups;
in acid form or in the form of any pharmaceutically acceptable salt thereof.
2. Compounds according to Claim 1, in which R2 represents a
monosaccharide of formula (II) and R7 represents a hydroxyl group.
3. Compounds according to Claim 1 or Claim 2, corresponding to
formula (1-1) below, in which R7 represents a hydroxyl group and R, R1, R4 and
R6 are as defined in Claim 1:

in acid form or in the form of any pharmaceutically acceptable salt thereof.
4. Compounds according to Claim 1, in which R2 represents a group
-O-alkyl.
5. Compounds according to Claim 1 or Claim 4, corresponding to
formula (I-2) below, in which R2 represents a group -O-alkyl and R1, R4, R6, R8
and R9 are as defined in Claim 1:

in acid form or in the form of any pharmaceutically acceptable salt thereof.
6. Compounds according to Claim 1, in which:
- R2 represents a group -O-alkyl, and
- R7 represents a disaccharide of formula (VI) as defined in Claim 1, in
which R9 represents a disaccharide of formula (VII) as defined in Claim 1.
7. Compounds according to Claim 1 or Claim 6, corresponding to
formula (I-3) below, in which R2 and Rg are as defined in Claim 6 and R1, R4, R6,
R8 and R10 are as defined in Claim 1:

in acid form or in the form of any pharmaceutically acceptable salt thereof.
8. Compounds according to Claim 1, in which:
- R2 represents a group -O-alkyl, and
- R7 represents a disaccharide of formula (VI) as defined in Claim 1, in
which R9 represents either a hydroxyl group or a group -O-alkyi.
9. Compounds according to Claim 1 or Claim 8, corresponding to
formula (I-2) below, in which R1, R4, R6 and R8 are as defined in Claim 1, R2
represents a group -O-alkyl and R9 represents either a hydroxyl group or a group
-O-alkyl:

in acid form or in the form of any pharmaceutically acceptable salt thereof.
10. Compounds according to Claim 8 or Claim 9, in which R9 represents a
group -O-alkyl.
11. Compounds according to any one of Claims 1 to 10, characterized in
that they correspond to the formula (I'), in which R1, R2, R4, R6 and R7 are as
defined in any one of Claims 1 to 9:

12. Compounds according to any one of Claims 1 to 11, chosen from the
following compounds:
- methyl (sodium 4-O-propyl-2-O-sodium sulfonato-a-L-idopyranosyl-
uronate)-(1?4)-(2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-
glucopyranosyl)-(1?4)-[(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-
(1?4)-(2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-gluco-
pyranosyl-(1?4)]2-(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-
(1?4)-2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-
glucopyranoside (No. 1);
- methyl (sodium 4-O-propyl-2-O-sodium sulfonato-a-L-idopyranosyl-
uronate)-(1?4)-(2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-
glucopyranosyl)-(1?4)-[(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-:
(1?4)-(2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-gluco-
pyranosyl-(1?4)]3-(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-
(1?4)-2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-gluco-
pyranoside (No. 2);
- sodium [methyl (sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-
(1?4)-(2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-
glucopyranosyl)-(1?4)-[(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-
(1?4)-(2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-
glucopyranosyl)-(1?4)]2-2-O-sodium sulfonato-a-L-idopyranoside]-uronate
(No. 3);
- methyl (sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-
(2-deoxy-2-sodium (sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-
sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-deoxy-2-sodium (sulfonato-
amino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-sodium sulfonato-a-L-
idopyranosyluronate)-(1?4)-(2-deoxy-6-O-sodium suifonato-2-sodium
(sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-sodium sulfonato-a-L-
idopyranosyluronate)-(1?4)-2-deoxy-6-O-sodium sulfonato-2-sodium
(sulfonatoamino)-a-D-glucopyranoside (No. 4);
- methyl (sodium 2-O-sodium suifonato-a-L-idopyranosyluronate)-(1?4)-
(2-deoxy-2-sodium (sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-
sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-deoxy-6-O-sodium
sulfonato-2-sodium (sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-
sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-deoxy-2-sodium
(sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-sodium sulfonato-a-L-
idopyranosyluronate)-(1?4)-2-deoxy-6-O-sodium sulfonato-2-sodium
(sulfonatoamino)-a-D-glucopyranoside (No. 5);
- methyl (sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-
(2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-glucopyranosyl)-
(1?4)-(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-deoxy-
2-sodium (sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-sodium
sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-deoxy-2-sodium (sulfonatoamino)-
a-D-glucopyranosyl)-(1?4)-(sodium 2-O-sodium sulfonato-a-L-idopyranosyl-
uronate)-(1?4)-2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-
giucopyranoside (No. 6);
- methyl (sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-
(2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-glucopyranosyl)-
(1?4)-(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-deoxy-
2-sodium (sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-sodium
sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-deoxy-6-O-sodium sulfonato-2-
sodium (sulfonatoamino)-a-D-glucopyranosyl)-(1?4)-(sodium 2-O-sodium
sulfonato-a-L-idopyranosyluronate)-(1?4)-2-deoxy-6-O-sodium sulfonato-2-
sodium (sulfonatoamino)-a-D-glucopyranoside (No. 7);
- methyl (sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-
(2-deoxy-6-O-sodium sulfonato-2-sodium(sulfonatoamino)-a-D-glucopyranosyl)-
[(1?4)-(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-deoxy-
6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-glucopyranosyl)]2-(1?4)-
(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-2-deoxy-6-O-
sodium sulfonato-2- (sulfonato)amino-a-D-glucopyranoside (No. 8); and
- methyl (sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-
(2-deoxy-6-O-sodium sulfonato-2-sodium (sulfonato)amino-a-D-glucopyranosyl)-
[(1?4)-(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-(2-deoxy-
6-O-sodium sulfonato-2-sodium (sulfonatoamino)-a-D-glucopyranosyl)]2-(1?4)-
(sodium 2-O-sodium sulfonato-a-L-idopyranosyluronate)-(1?4)-2-deoxy-2-
sodium (sulfonatoamino)-a-D-glucopyranoside (No. 9).
13. Medicament, characterized in that it comprises a compound according
to any one of Claims 1 to 12 and in which R7 represents either a hydroxyl group
or a monosaccharide of formula (V) below, or a disaccharide of formula (VI), in
which R8 and R9 are as defined in any one of Claims 1 to 10:

or a pharmaceutically acceptable salt thereof.
14. Medicament according to Claim 13, characterized in that it comprises
a compound according to any one of Claims 1 to 12, or a pharmaceutically
acceptable salt thereof.
15. Medicament according to Claim 13, characterized in that it comprises
compound 10:

or a pharmaceutically acceptable salt thereof.
16. Pharmaceutical composition, characterized in that it comprises a
compound according to any one of Claims 1 to 12, and in which R7 represents
either a hydroxyl group or a monosaccharide of formula (V) below, or a
disaccharide of formula (VI), in which R8 and R9 are as defined in any one of
Claims 1 to 10:

or a pharmaceutically acceptable salt thereof, and also at least one
pharmaceutically acceptable excipient.
17. Pharmaceutical composition, characterized in that it comprises a
compound according to any one of Claims 1 to 12, or a pharmaceutically
acceptable salt thereof, and also at least one pharmaceutically acceptable
excipient.
18. Pharmaceutical composition, characterized in that it comprises
compound 10:
or a pharmaceutically acceptable salt thereof, and also at least one
pharmaceutically acceptable excipient.
19. Compound according to any one of Claims 1 to 12, in which R7
represents either a hydroxyl group or a monosaccharide of formula (V) below, or
a disaccharide of formula (VI), in which R8 and R9 are as defined in any one of
Claims 1 to 10:

for its use in the treatment of pathologies requiring activation of the FGF
receptors.
20. Compound according to any one of Claims 1 to 12, or
pharmaceutically acceptable salt thereof, for its use in the treatment of
pathologies requiring activation of the FGF receptors.
21. Compound 10, or a pharmaceutically acceptable salt thereof:
for its use in the treatment of pathologies requiring activation of the FGF
receptors.
22. Compound according to any one of Claims 19 to 21, for its use in the
treatment of pathologies requiring angiogenesis activation and post-ischaemic
revascularization.
23. Compound according to any one of Claims 19 to 22, for its use in the
treatment of ischaemia, such as cardiac ischaemia and arterial ischaemia of the
lower limbs, the treatment of diseases associated with narrowing or obstruction
of the arteries or arterites, the treatment of angina pectoris, the treatment of
thromboangitis obliterans, the treatment of atherosclerosis, the treatment of
inhibition of restenosis after angioplasty or endoarterectomy, the treatment of
cicatrization, muscle regeneration treatment, treatment for the survival of
myoblasts, the treatment of peripheral neuropathy, the treatment of post-
operative nerve damage, the treatment of nerve deficiencies such as
Parkinson's disease, Alzheimer's disease, prion disease and neuronal
degeneration in alcoholics, the treatment of dementias, treatment for improving
the survival of a bioartificial pancreas graft in the case of diabetics, treatment for
improving the revascularization of grafts and the survival of grafts, the treatment
of retinal degeneration, the treatment of pigmentary retinitis, the treatment of
osteoarthritis, the treatment of pre-eclampsia or the treatment of vascular lesions
and of acute respiratory distress syndrome, treatment for repairing cartilage,
treatment for repairing and protecting bones, treatment for repairing and
protecting hair follicles and for protecting and regulating hair growth.
24. Compound of formula 20A, in which Pg, Pg' and Pg", which may be
identical or different, represent protecting groups:
25. Compound according to Claim 24, in which Pg, Pg' and Pg" represent,
respectively, benzyl, allyl and acetyl groups.

ABSTRACT

The invention relates to FGF
receptor-activating N-sulfate
oligosaccharides having Formula (I),
wherein R1, R4, R6, and R2 are -OSO3- or
hydroxyl groups, R2 is an -O-alkyl group or a
monosaccharide having Formula (II), R3 is a
disaccharide having Formula (III), R3 is a
disaccharide having Formula (IV), R7 is a
hydroxyl group or a disaccharide having
Formula (VI), and R9 is a hydroxyl or -O-
alkyl group or a disaccharide having Formula
(VII), where R10 is an -O-alkyl group. The
invention further relates to the preparation of
said oligosaccharides and to the therapeutic
use thereof