U.S. patent application number 13/681132 was filed with the patent office on 2013-07-11 for use of modified oligo-b-(1,3)-glucanes for treating diseases of the immune system oligo-b-(1,3v-glucane-(1,3)-mannose, oligo-b-(1,3)-glucane-(1,3)-mannitol and derivatives thereof, methods for preparing the same and drugs containing them.
This patent application is currently assigned to ASE & BIO.. The applicant listed for this patent is ASE & BIO.. Invention is credited to Karine DESCROIX, Vincent FERRIERES, Frank JAMOIS, Isabelle LAURENT, Vaclav VETVICKA, Jean-Claude YVIN.
Application Number | 20130178616 13/681132 |
Document ID | / |
Family ID | 37896020 |
Filed Date | 2013-07-11 |
United States Patent
Application |
20130178616 |
Kind Code |
A1 |
YVIN; Jean-Claude ; et
al. |
July 11, 2013 |
USE OF MODIFIED OLIGO-B-(1,3)-GLUCANES FOR TREATING DISEASES OF THE
IMMUNE SYSTEM OLIGO-B-(1,3V-GLUCANE-(1,3)-MANNOSE,
OLIGO-B-(1,3)-GLUCANE-(1,3)-MANNITOL AND DERIVATIVES THEREOF,
METHODS FOR PREPARING THE SAME AND DRUGS CONTAINING THEM
Abstract
The present invention relates to the use of at least one
compound of formula (III), (IV), (V) or (VI), in which p is an
integer from 0 to 9; and R2 represents hydrogen, allyl,
methylnaphthyl, benzyl, paramethoxybenzyl, or halogenoacetyl, for
the preparation of a medicament for treating diseases such as
tumors, cancer, viral disease, bacterial disease, fungal disease,
disease of the immune system, auto-immune disease or disease linked
to a deficiency in immunostimulation, in human beings and
warm-blooded animals. The invention also relates to new products
having a mannose or mannitol termination as well as a method for
preparing them.
Inventors: |
YVIN; Jean-Claude; (Saint
Malo, FR) ; DESCROIX; Karine; (Saint-colomban,
FR) ; FERRIERES; Vincent; (Gahard, FR) ;
JAMOIS; Frank; (La Chapelle Des Fouegeretz, FR) ;
LAURENT; Isabelle; (Rennes, FR) ; VETVICKA;
Vaclav; (Louisville, KY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ASE & BIO.; |
Paris |
|
FR |
|
|
Assignee: |
ASE & BIO.
Paris
FR
|
Family ID: |
37896020 |
Appl. No.: |
13/681132 |
Filed: |
November 19, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12520612 |
Mar 12, 2010 |
8367641 |
|
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PCT/FR07/52595 |
Dec 20, 2007 |
|
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13681132 |
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Current U.S.
Class: |
536/123.1 |
Current CPC
Class: |
C07H 3/06 20130101; A61P
37/02 20180101; A61P 35/00 20180101; A61P 31/04 20180101; Y02P
20/55 20151101; A61K 31/702 20130101; A61P 31/12 20180101; A61P
37/04 20180101; C07H 3/08 20130101; A61P 31/10 20180101; A61P 37/06
20180101 |
Class at
Publication: |
536/123.1 |
International
Class: |
C07H 3/06 20060101
C07H003/06 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 2006 |
FR |
06 11333 |
Claims
1. A compound selected from the group consisting of formulae (III),
(IV), (V) and (VI) below: ##STR00038## wherein: p is an integer
from 0 to 9; R2 represents hydrogen, allyl, methylnaphthyl, benzyl,
paramethoxybenzyl, or halogenoacetyl; R3 and R4, on the one hand,
and R'3 and R'4, on the other hand, together form an ethylidyl,
isopropylidyl, hexafluoroisopropylidyl, cyclopentylidyl,
cyclohexylidyl, cycloheptylidyl, butylidyl, 1-tertiobutylethylidyl,
benzylidyl, methoxybenzylidyl, or 1-phenylbenzylidyl radical, or
R3, R4, R3' and R4' represent each independently of each other a
benzyl, chlorobenzyl, nitrobenzyl, allyl, triarylmethyl, or
trialkylsilyl, ester; R5 represents H, a levulinoyl, acetyl,
chloroacetyl, fluorenylmethyloxycarbonyl, or trialkylsilyl group,
provided that none of R3, R4, R3', R4' is identical to R5; R7
represents an ester; R8, a group orthogonal to all the other
protective groups, which can be selected from the group consisting
of Bn, NAP, p-methoxybenzyl, p-methoxyphenyl, and allyl, and R9
represents OR8 or X.
2. The compound according to claim 1, wherein p is an integer from
0 to 6.
3. The compound according to claim 1, wherein p is equal to 2, 3 or
4.
4. The compound according to claim 1, wherein the halogenoacetyl
group of R2 is selected from the group consisting of chloroacetyl,
bromoacetyl and iodoacetyl.
5. The compound according to claim 1, wherein the trialkylsilyl
group of R3, R4, R3', R4' and R5 is selected from the group
consisting of triethylsilyl, tri-iso-propylsilyl and
tertiobutyldimethylsilyl.
6. The compound according to claim 1, wherein the ester group of
R3, R4, R3' and R4' is selected from the group consisting of
acetyl, chloroacetyl, benzoyl, and pivaloyl.
7. The compound according to claim 1, wherein R5 represents a
levulinoyl group.
8. The compound according to claim 1, wherein the ester group of R7
is selected from the group consisting of acetyl, benzoyl, and
pivaloyl.
9. The compound according to claim 1, wherein the ester group of R7
represents a benzoyl group.
10. The compound according to claim 1, wherein R8 represents Bn.
Description
[0001] This application is a Divisional of U.S. application Ser.
No. 12/520,612, filed Mar. 12, 2010, which is a 371 application of
PCT/FR2007/052595, filed Dec. 20, 2007, all of said applications
incorporated herein by reference.
[0002] The present invention relates to the use of modified
oligo-.beta.-(1,3)-glucans of formula (I) or (II) below for the
preparation of medicaments which are useful in treatments based on
the stimulation of the immune system.
[0003] The glucans, which are natural products, have long been
studied and the immunostimulant properties of certain glucans are
known. However, not all the natural glucans are active.
[0004] The applicant company has shown, in particular in
WO03/045414 that Laminarine, which is a polysaccharide extracted
from the brown alga Laminaria digitata has immunostimulation
activities. Laminarine is a polysaccharide with a low molecular
weight constituted by a main linear .beta.-(1,3)-glucan chain, of
20 to 30 glucose units weakly branched in position 6, which has at
the terminal end either a glucose entity (G chain) or a mannitol
entity (M chain).
[0005] Although the immunostimulant activity of Laminarine has been
recognized, a major drawback of its use for therapeutic purposes is
its natural origin. In fact, the composition of Laminarine is not
constant and depends in particular on where and when it is
collected, etc.
[0006] A real need therefore exists for synthetic products having
the therapeutic properties of Laminarine.
[0007] The present inventors have turned more particularly to the M
group oligosaccharides present in Laminarine. In the literature, it
is mentioned that these oligosaccharides have a bond of
configuration between carbon number 1 of the terminal entity of the
glucan and a primary carbon of the mannitol residue, and can be
represented as follows:
##STR00001##
[0008] They have sought to synthesize these molecules in order to
provide in particular the pharmaceutical industry with molecules
the characterization of which is complete and the structure
completely clarified, thus avoiding the difficulties inherent in
the use of natural products as pharmaceutical products.
[0009] Unexpectedly and surprisingly, the present inventors found
that the oligosaccharides of formula (I) or (II) below, which
themselves have a .beta. bond between carbon number 1 of the
terminal glucose entity and a secondary carbon of the mannose or
mannitol terminal entity, had immunostimulant activities.
[0010] A first subject of the invention therefore relates to the
use of at least one modified oligo-.beta.-(1,3)-glucan of formula
(I) or (II)
##STR00002##
in which R.sub.1 represents H or OH and n is an integer from 2 to
10, preferably from 2 to 7, and still more preferably equal to 3, 4
or 5,
[0011] for the preparation of a composition intended for the
treatment of diseases chosen from the group including tumour,
cancer, viral disease, bacterial disease, fungal disease, disease
of the immune system, auto-immune disease or disease linked to a
deficiency in immunostimulation, in humans and warm-blooded
animals. The action of the compounds of formula (I) or (II) is
linked to the presence of receptors specific to these compounds at
the surface of the macrophages and more generally of the different
populations of leucocytes. This activity is attributed to the
stimulation of immunocompetent cells of the organism and is
measured via: [0012] the activation of the NK cells, T lymphocytes,
and nuclear factor .kappa.B (NF-.kappa.B); [0013] the phagocytic
activity; [0014] the secretion of cytokines such as the
interleukins (IT), TNF-.alpha., or also interferon .gamma.; [0015]
the production of reactive oxygen species such as the superoxide
anion or hydrogen peroxide.
[0016] Advantageously, when in the compound of formula (II),
R.sub.1 represents OH, the terminal group is a mannitol.
[0017] The interaction mechanisms of numerous glucans have been
broadly described, but have shown results which are often
contradictory, making it impossible to predict which glucan is the
best immunomodulator. As for the effects linked to functional or
structural modifications, these are completely unpredictable.
[0018] According to the invention, the composition used according
to the invention can be administered to the patient by intravenous,
intraperitoneal or oral route.
[0019] The doses of compounds of formula (I'), (II') are a function
of the pathology to be treated and the adopted administration
method. They are generally from 25 to 70 IU/kg of bodyweight per
day, more preferably from 35 to 45 IU/kg of bodyweight per day.
[0020] Useful medicaments which are administered by oral route are
tablets, granules, syrup, gelatin capsules, gel.
[0021] According to another embodiment, the composition used
according to the invention can also comprise a chemotherapeutic
agent.
[0022] The chemotherapeutic agent is chosen from the group
comprising cisplatin, vinblastine, paclitaxel, taxol and its
derivatives, monoclonal antibodies such as in particular Rituximab
and Cituximab.
[0023] When the composition comprises a chemotherapeutic agent
and/or a potentiator, according to the invention, it can be used in
a sequenced manner, i.e. the compound of formula (I) or (II) is
administered at a certain point in time and the chemotherapeutic
agent is administered at another point in time optionally with the
potentiator.
[0024] The present invention also relates to certain derivatives of
formula (I) and (II) above which are novel compounds. These novel
compounds are oligo-.beta.-(1,3) glucan-(1,3)-mannose and
oligo-.beta.-(1,3) glucan-(1,3)-mannitol and their deoxy
derivatives in position 4, which have the general formula (I') or
the general formula (II') respectively:
##STR00003##
in which R.sub.1 represents H or OH and n is an integer from 2 to
10, preferably from 2 to 7, and still more preferably equal to 3, 4
or 5. More particularly, such compounds are the following
compounds: [0025]
.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-(.alpha.,.beta.)-D-mannopyranose; [0026]
.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-mannopyranose;
[0027]
.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-4-deoxy-(.alpha.,.beta.)-D-deoxy-mannopyranose; [0028]
.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-mannitol; [0029]
.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-4-deoxy-mannitol; [0030]
.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-.beta.-D-glucopyranosyl-(1.fwdarw.3)-mannitol; [0031]
.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-.beta.-D-glucopyranosyl-(1.fwdarw.3)-4-deoxy-(.alpha.,.beta.)-D-mannopyra-
nose; [0032]
.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-.beta.-D-glucopyranosyl-(1.fwdarw.3)-4-deoxy-mannitol.
[0033] These novel compounds are useful as pharmaceutical
compounds.
[0034] A subject of the invention is therefore also medicaments
comprising these novel compounds with at least one excipient chosen
as a function of the adopted administration method and dosage.
[0035] These medicaments can be medicaments intended for
administration by oral route, by intravenous route or by
intraperitoneal route.
[0036] The doses of compounds of formula (I'), (II') are a function
of the pathology to be treated and of the adopted method of
administration. They are generally from 25 to 70 IU/kg of
bodyweight per day, more preferably from 35 to 45 IU/kg of
bodyweight per day.
[0037] Useful medicaments administered by oral route are tablets,
granules, syrup, gelatin capsules, gel.
[0038] These medicaments can also comprise at least one
chemotherapeutic agent chosen from the group comprising cisplatin,
vinblastine, paclitaxel, taxol and its derivatives, monoclonal
antibodies such as in particular Rituximab and Cituximab.
[0039] In particular, these compounds are useful as
immunomodulatory agents.
[0040] Another subject of the invention relates to the use of at
least one compound of formula (I') or (II') for the treatment of a
disease chosen from the group including tumour, cancer, viral
disease, bacterial disease, fungal disease, disease of the immune
system, auto-immune disease or disease linked to a deficiency in
immunostimulation, in warm-blooded animals and human beings.
[0041] These compounds have an original structure since the
glucanic part is linked to the mannitol residue by one of these
secondary hydroxyls via a bond of configuration .beta.. The
presence of the molecules of formula (I') and (II') in which
R.sub.1 represents --OH has been detected in Laminaria digita. No
preparation method has been described.
[0042] The preparation by chemical route of the novel compounds of
formula (I') or (H') is based mainly on epimerization of the
D-glucose type terminal reducing entity to D-mannose type residue.
This involves the singularization of the reducing entity with
respect to the other glucose units of the oligossaccharide and the
singularization of the hydroxyl 2 of this entity with respect to
the other functions of the reducing unit. Two different synthesis
routes are envisaged, an iterative synthesis and a synthesis by
epimerization of the terminal entity.
[0043] According to the first embodiment, the method for the
preparation of the novel compounds comprises a reaction between a
glycosyl donor of formula (D) below and a glycosyl acceptor of
formula (A) below:
##STR00004##
in which m is an integer from 1 to 9, preferably from 1 to 6, and
still more preferably equal to 2, 3 or 4; p is an integer from 0 to
9, preferably from 0 to 6, and still more preferably equal to 2, 3
or 4; R represents alkyl such as ethyl, methyl, propyl or butyl, or
aryl such as phenyl or tolyl; R.sub.2 represents allyl,
methylnaphthyl, benzyl, paramethoxybenzyl, halogenoacetyl
(chloroacetyl, bromoacetyl, iodoacetyl); R.sub.3 and R.sub.4, on
the one hand, and R.sub.3' and R.sub.4', on the other hand,
together form an ethylidyl, isopropylidyl, hexafluoroisopropylidyl,
cyclopentylidyl, cyclohexylidyl, cycloheptylidyl, butylidyl,
1-tertiobutylethylidyl, benzylidyl, methoxybenzylidyl,
1-phenylbenzylidyl radical or R.sub.3, R.sub.4, R.sub.3' and
R.sub.4' each represent independently of each other a benzyl,
chlorobenzyl, nitrobenzyl, allyl, triarylmethyl, trialkylsilyl such
as triethylsilyl, tri-isopropylsilyl, tertiobutyldimethylsilyl,
ester such as acetyl, chloroacetyl, benzoyl, pivaloyl; R.sub.5
represents H, a levulinoyl, acetyl, chloroacetyl,
fluorenylmethyloxycarbonyl, trialkylsilyl group such as
triethylsilyl, tri-isopropylsilyl, tertiobutyldimethylsilyl,
preferably a levulinoyl group, provided that none of R.sub.3,
R.sub.4, R.sub.3', R.sub.4' is identical to R.sub.5.
[0044] In the present application the following abbreviations are
used:
Bn: benzyl, NAP: 2-methylnaphthyl, Bz: benzoyl, X: halogen, SEt:
Ethylthio-, Ac: acetyl, Lev: levulinoyl.
[0045] Advantageously, the compound of formula (A) is
##STR00005##
Advantageously, the compound of formula (D) is
##STR00006##
[0046] This stage of the method can be included in an iterative
synthesis which differs from that described in FR2804684 by the
necessary use of a first acceptor comprising in position 2 a
protective group orthogonal to all the other groups present on the
oligomers synthesized after elongation of the chain, which makes
regioselective epimerization possible.
[0047] This iterative synthesis can be described according to the
reaction diagram (reaction diagram 1) as given hereafter:
##STR00007##
[0048] Reaction diagram 1 above comprises in the following order:
[0049] the preparation [a'] from a compound 1 as obtained in
FR2804694, of a compound 2 the --SEt group of which has been
replaced by an --OBn group; [0050] the substitution [a] in position
2 of --OBz by --OR.sub.5, R.sub.5 preferably being levulinoyl, and
the selective deprotection [b] of the hydroxyl in position 3;
[0051] the reaction between the donor 1 and the acceptor 4 [c], or
glycosylation; [0052] optionally the selective deprotection [d] in
position 3 of the hydroxyl and the glycosylation between compound 1
and compound 6, these two stages being able to be reiterated until
the desired number n of glucose units is obtained; [0053] selective
cleavage [e] of the levulinoyl (Lev) group; advantageously this
cleavage is carried out with NH.sub.2NH.sub.2 in AcOH. [0054]
oxidation [f], preferably with Dess-Martin periodinane; [0055]
epimerizing reduction [g]; advantageously with L-selectride; [0056]
deprotection [h] of all the ester groups, advantageously with
MeONa/MeOH; [0057] cleavage [i] of all the hydrogenolyzable groups,
advantageously with H.sub.2, Pd(OAc).sub.2; [0058] optionally
reduction [j] of the mannose to corresponding mannitol,
advantageously with NaBH.sub.4 in MeOH.
[0059] It is also possible to envisage the synthesis by utilizing a
more convergent reaction diagram, as defined hereafter (reaction
diagram 2).
In this reaction diagram, the donor D is a disaccharide, or a
higher oligosaccharide, which is reacted with an acceptor A which
is also a disaccharide or which is a higher oligosaccharide: tri-,
tetra-, penta-, as obtained as compound 6 according to reaction
diagram 1.
##STR00008##
[0060] In this second reaction diagram, preferably R' is Ac, Bz or
pivaloyl and R.sub.5 is Lev.
[0061] This second reaction diagram comprises in the following
order: [0062] the reaction [a''] between a glycosyl donor
disaccharide (20) as obtained in FR2804694, and a glycosyl acceptor
as prepared according to the first reaction diagram, advantageously
in the presence of TMSOTf (trifluoromethanesulphonic acid); [0063]
selective cleavage [b''] of the levulinoyl (Lev) group,
advantageously with NH.sub.2NH.sub.2 in AcOH; [0064] Oxidation
[c''], advantageously with Dess-Martin peridionane; [0065]
epimerizing reduction [d''], advantageously with L-selectride;
[0066] complete deprotection [e''], advantageously by MeONa/MeOH,
reaction followed by H.sub.2, Pd(OAc).sub.2.
[0067] According to a second embodiment, the method for preparing
the novel compounds comprises a stage of epimerization in position
2 of the terminal member. This synthesis route also makes it
possible to obtain the deoxygenated compounds in position 4.
[0068] Reaction diagram 3 below illustrates this embodiment:
##STR00009## ##STR00010##
[0069] In reaction diagram 3, R.sub.7 is an ester, in particular
acetyl, benzoyl, pivaloyl, preferably Bz, and R.sub.8, a group
orthogonal to all the other protective groups, can be Bn, NAP,
p-methoxybenzyl, p-methoxyphenyl, allyl, and preferably Bn, X is
preferably a bromine.
[0070] This third reaction diagram comprises in the following
order: [0071] the protection reaction [A] of all the OH groups,
preferably the protective group is Bz and the reaction is then
carried out with BzCl in pyridine, [0072] selective halogenation
[B], advantageously bromination with HBr in AcOH; [0073]
elimination [C], advantageously with DBU diazabicycloundecene;
[0074] addition [D], advantageously with N-bromosuccinimide in MeOH
[0075] substitution in the anomeric position and elimination of the
protected hydroxy group in position 4 of the terminal entity [E],
advantageously with Ph.sub.3PO or a Lewis acid such as silver
triflate in benzyl alcohol or any other alcohol including
naphthalenemethanol, p-methoxybenzyl alcohol, p-methoxyphenol,
allyl alcohol; [0076] epimerizing reduction [F], advantageously
with L-selectride; [0077] selective deprotection [G] of the R.sub.7
protective groups, advantageously in MeONa/MeOH; [0078] selective
deprotection [H] of the R.sub.8 protective group, advantageously
with H.sub.2Pd(OAc).sub.2 when the group is hydrogenolyzable;
[0079] reduction [I] of deoxy-mannose to deoxy-mannitol,
advantageously with NaBH.sub.4 in EtOH/H.sub.2O.
[0080] The synthesis intermediates used in the methods described
above are novel products. A subject of the invention is therefore
also the compounds of formulae (III), (IV), (V) and (VI):
##STR00011##
in which: p is an integer from 0 to 9, preferably from 0 to 6, and
still more preferably equal to 2, 3 or 4; R.sub.2 represents
hydrogen, allyl, methylnaphthyl, benzyl, paramethoxybenzyl,
halogenoacetyl (chloroacetyl, bromoacetyl, iodoacetyl); R.sub.3 and
R.sub.4, on the one hand, and R'.sub.3 and R'.sub.4, on the other
hand, together form an ethylidyl, isopropylidyl,
hexafluoroisopropylidyl, cyclopentylidyl, cyclohexylidyl,
cycloheptylidyl, butylidyl, 1-tertiobutylethylidyl, benzylidyl,
methoxybenzylidyl, 1-phenylbenzylidyl radical, or R.sub.3, R.sub.4,
R.sub.3' and R.sub.4'each represent independently of each other a
benzyl, chlorobenzyl, nitrobenzyl, allyl, triarylmethyl,
trialkylsilyl such as triethylsilyl, tri-isopropylsilyl,
tertiobutyldimethylsilyl, ester such as acetyl, chloroacetyl,
benzoyl, pivaloyl; R.sub.5 represents H, a levulinoyl, acetyl,
chloroacetyl, fluorenylmethyloxycarbonyl, trialkylsilyl group such
as triethylsilyl, tri-isopropylsilyl, tertiobutyldimethylsilyl,
preferably a levulinoyl group, provided that none of R.sub.3,
R.sub.4, R.sub.3', R.sub.4' is identical to R.sub.5; R.sub.7
represents an ester, in particular acetyl, benzoyl, pivaloyl,
preferably Bz; R.sub.8, a group orthogonal to all the other
protective groups, which can be chosen from Bn, NAP,
p-methoxybenzyl, p-methoxyphenyl, allyl, and preferably R.sub.8
represents Bn, R.sub.9 represents OR.sub.8 or X.
[0081] The compounds of formula (III) are acceptor compounds A as
defined previously.
[0082] The invention will be still better understood with the aid
of the remainder of the description which follows and examples
which are in no way limitative but correspond to advantageous
embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0083] FIG. 1 is a graphic representation of the average results of
the tests conducted in Example 8.
[0084] FIG. 2 is a graphic representation of the average results of
the tests conducted in Example 9.
[0085] FIG. 3 is a graphic representation of the average results of
the tests conducted in Example 10.
[0086] FIG. 4 is a graphic representation of the average results of
the tests conducted in Example 11.
EXAMPLES
[0087] Examples 1 to 7 illustrate the methods for synthesis of the
products of the invention, and Examples 8 to 11 show the biological
activity of the compounds of the invention.
Example 1
Preparation according to reaction diagram 1 of
.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-(.alpha.,.beta.)-D-mannopyranose (product A1)
[0088] Products 1 and 2 are prepared according to the method
described in FR 2 804 684 and Jamois, F.; Ferrieres, V.; Guegan,
J.-P.; Yvin, J.-C.; Plusquellec, D.; Vetvicka, V. Glycobiology
2005, 15, 393-407.
[a]--Preparation of benzyl
4,6-O-benzylidene-3-O-(2-methylnaphthyl)-2-O-levulinoyl-.beta.-D-glucopyr-
anoside of (product 3)
##STR00012##
[0090] Compound 2 (1 g, 2.006 mmol) is put into solution in 50 mL
of dichloromethane then DMAP (50 mg, 4.093 mmol), levulinoic acid
(250 .mu.L, 2.441 mmol) and DCC (500 mg, 2.423 mmol) are added.
After stirring for 5 hours at ambient temperature, the DCU is
eliminated by filtration, then the medium is taken up in 50 mL of
dichloromethane and washed with an aqueous solution of 10%
hydrochloric acid, a saturated aqueous solution of sodium
bicarbonate and a saturated aqueous solution of sodium chloride.
After drying the organic phase over MgSO.sub.4 and concentration,
the compound is purified on silica gel [petroleum ether/ethyl
acetate (3:1; v/v)] in order to yield in a quantitative manner 1.2
g of the sought compound 3.
[0091] Product 3: white solid; MP (.degree. C.) 133; Rf (EP/AcOEt,
3:1) 0.4; [.alpha.].sub.D.sup.20 -37.4 (c=1.0,
CH.sub.2Cl.sub.2).
[0092] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 7.83-7.74
(m, 4H, arom. H); 7.53-7.28 (m, 13H, arom. H); 5.61 (s, 1H, H7);
5.01 (d, 1H, H9, J.sub.H9-H9'=12.4 Hz); 4.87 (d, 1H, H9'); 4.86 (d,
1H, H8, J.sub.H8-H8'=12.4 Hz); 4.60 (d, 1H, H8'); 2.58 (t, 2H, H12,
J.sub.H11-H12=6.6 Hz); 2.44 (t, 2H, H11); 2.09 (s, 3H, H14) and
Table 1a.
[0093] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 206.2
(C13); 171.3 (C10); 137.1, 136.9, 135.6, 133.1, 132.9 (5C, quat.
arom. C); 129.0, 128.4, 128.3, 128.2, 128.0, 127.9, 127.8, 127.7,
127.6, 126.8, 126.1, 126.0, 125.8 (arom. C); 101.3 (C7); 74.1 (C9);
70.7 (C8); 37.7 (C12); 29.8 (C14); 27.8 (C11) and Table 1b.
[0094] Ultimate analysis (C.sub.36H.sub.36O.sub.8): Theoretical:
C=72.47%, H=6.08%; Measured: C=72.05%, H=6.12%.
[0095] HRMS (ESI.sup.+): [M+Na].sup.+ C.sub.36H.sub.36NaO.sub.8:
theoretical m/z: 619.2308, measured m/z: 619.2308; [M+K].sup.+
C.sub.36H.sub.36KO.sub.8: theoretical m/z: 635.2047, measured m/z:
635.2050.
[b] Preparation of benzyl
4,6-O-benzylidene-2-O-levulinoyl-.beta.-D-glucopyranoside (Product
4)
##STR00013##
[0097] The monosaccharide 3 (1.1 g, 1.844 mmol) and
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (810 mg, 3.568
mmol) are introduced into 50 mL of a dichloromethane/methanol
mixture (4:1; v/v) at ambient temperature. After reaction for 5
hour, the medium is diluted with 50 mL of dichloromethane and
washed twice with a saturated aqueous solution of sodium
bicarbonate. After drying (MgSO.sub.4) and concentration of the
organic phase, the residue is purified on silica gel [petroleum
ether/ethyl acetate (3:1; v/v)]. In this way 690 mg of the sought
product 4 is obtained with a yield of 82%.
[0098] Product 4: white solid; MP (.degree. C.) 122; Rf (EP/AcOEt,
3:1) 0.2; [.alpha.].sub.D.sup.20 -62.2 (c=1.0,
CH.sub.2Cl.sub.2).
[0099] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 7.50-7.48
(m, 2H, arom. H); 7.38-7.28 (m, 8H, arom. H); 5.55 (s, 1H, H7);
4.89 (d, 1H, H8, J.sub.H8-H8'=12.3 Hz); 4.62 (d, 1H, H8'); 3.05 (s,
1H, OH3); 2.79-2.73 (m, 2H, H11); 2.59-2.51 (m, 2H, H10); 2.09 (s,
3H, H14); 2.16 (s, 3H, H13) and Table 1a.
[0100] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 207.1
(C12); 172.0 (C9); 136.9 (2C, quat. arom. C); 129.2, 128.4, 128.3,
127.9, 127.7, 126.3 (arom. C); 101.9 (C7); 70.8 (C8); 38.1 (C11);
29.8 (C13); 28.0 (C10) and Table 1b.
[0101] Ultimate analysis (C.sub.25H.sub.28O.sub.8): Theoretical:
C=65.78%, H=6.18%; Measured: C=66.04%, H=6.23%.
[0102] HRMS (ESI.sup.+): [M+Na].sup.+ C.sub.25H.sub.28NaO.sub.8:
theoretical m/z: 479.1682, measured m/z: 479.1682; [M+K].sup.+
C.sub.25H.sub.28KO.sub.8: theoretical m/z: 495.1421, measured m/z:
495.1395.
[c] Preparation of benzyl
2-O-benzoyl-4,6-O-benzylidene-3-O-(2-methylnaphthyl)-.beta.-D-glucopyrano-
syl-(1.fwdarw.3)-4,6-O-benzylidene-2-O-levulinoyl-.beta.-D-glucopyranoside
(Product 5)
##STR00014##
[0104] The donor 1 (1.73 g, 3.108 mmol) and the acceptor 4 (1.29 g,
2.826 mmol) are introduced into anhydrous dichloromethane in a
flask at -60.degree. C., in the presence of a 4 .ANG. molecular
sieve. N-iodosuccinimide (NIS) (763 mg, 3.390 mmol) and
trimethylsilyl triflate (50 .mu.L, 0.283 mmol) are then added to
the medium. After reaction for 3 hours, the medium is neutralized
with triethylamine, filtered then concentrated. After purification
by silica gel chromatography [toluene/ethyl acetate (9:1; v/v)],
2.51 g of the sought product 5 is obtained with a yield of 94%.
[0105] Product 5: white solid; MP (.degree. C.) 178-179; Rf
(EP/AcOEt, 3:1) 0.2; [.alpha.].sub.D.sup.20 -21.0 (c=1.0,
CH.sub.2Cl.sub.2).
[0106] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 7.90-7.86
(m, 2H, arom. H); 7.70-7.67 (m, 1H, arom. H); 7.60-7.21 (m, 24H,
arom. H); 5.53 (s, 1H, H7b); 5.28 (s, 1H, H7a); 4.94 (d, 1H, H8b,
J.sub.H8b-H8'b=12.1 Hz); 4.84 (d, 1H, H8'b); 4.80 (d, 1H, H8a,
J.sub.H8a-H8'a=12.2 Hz); 4.52 (d, 1H, H8'a); 2.66-2.46 (m, 2H,
H10a); 2.40-2.27 (m, 2H, H11a); 2.08 (s, 3H, H13a) and Table
2a.
[0107] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 206.4
(C12a); 171.1 (C9a); 165.0 (OCOPh); 137.3, 137.0, 136.7, 135.3,
133.1, 133.0 (7C, quat. arom. C); 132.8, 129.7, 129.4, 128.9,
128.3, 128.2, 127.9, 127.8, 127.7, 127.5, 126.7, 126.1, 126.0,
125.8, 125.6 (arom. C); 101.7 (C7b); 100.9 (C7a); 73.4 (C8b); 70.6
(C8a); 37.8 (C11a); 29.7 (C13a); 27.5 (C10a) and Table 2b.
[0108] Ultimate analysis (C.sub.56H.sub.54O.sub.14): Theoretical:
C=70.72%, H=5.72%; Measured: C=70.89%, H=5.72%.
[0109] HRMS (ESI.sup.+): [M+Na].sup.+ C.sub.56H.sub.54NaO.sub.14,
theoretical m/z: 973.3411; measured m/z: 973.3412.
[d]--Preparation of benzyl
2-O-benzoyl-4,6-O-benzylidene-.beta.-D-glucopyranosyl-(1.fwdarw.3)-4,6-O--
benzylidene-2-O-levulinoyl-.beta.-D-glucopyranoside (Product 6)
[0110] 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (820 mg, 3.600
mmol) is added to a solution of compound 5 (1.71 g, 1.800 mmol) in
a dichloromethane/methanol mixture (4:1; v/v). After reaction for
16 hours, the medium is diluted with dichloromethane, washed with a
saturated solution of sodium bicarbonate and water. The organic
phase is dried (MgSO.sub.4), evaporated and the sought product 6 is
obtained after purification on silica gel [petroleum ether/ethyl
acetate (2:1; v/v)] (1.325 g, yield=91%).
[0111] Product 6: white solid; MP (.degree. C.) 214; Rf (EP/AcOEt,
2:1) 0.3; [.alpha.].sub.D.sup.20 -43.0 (c=1.0,
CH.sub.2Cl.sub.2).
[0112] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 8.06-8.01
(m, 2H, arom. H); 7.63-7.23 (m, 19H, arom. H); 5.57 (s, 1H, H7b);
5.31 (s, 1H, H7a); 4.83 (d, 1H, H8a, J.sub.H8a-H8'a=12.2 Hz); 4.55
(d, 1H, H8'a); 2.72 (s, 1H, OH3b); 2.65-2.49 (m, 2H, H10a);
2.37-2.25 (m, 2H, H11a); 2.12 (s, 3H, H13a) and Table 2a.
[0113] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 206.4
(C12a); 171.1 (C9a); 165.7 (OCOPh); 137.0, 136.9, 136.7 (3C, quat.
arom. C); 133.3, 129.8 (arom. C); 129.5 (quat. arom. C); 129.3,
129.1, 128.4, 128.3, 128.2, 128.1, 127.8, 127.6, 126.2, 126.0
(arom. C); 101.6 (C7b); 101.5 (C7a); 70.6 (C8a); 37.7 (C11a); 29.7
(C13a); 27.5 (C10a) and Table 2b.
[0114] Ultimate analysis (C.sub.45H.sub.46O.sub.14): Theoretical:
C=66.66%, H=5.72%; Measured: C=66.53%, H=5.80%.
[0115] HRMS (ESI.sup.+): [M+Na].sup.+ C.sub.45H.sub.46NaO.sub.14:
theoretical m/z: 833.2785; measured m/z: 833.2785; [M+K].sup.+
C.sub.45H.sub.46KO.sub.14: theoretical m/z: 849.2525, measured m/z:
849.2530.
[c]--Preparation of benzyl
2-O-benzoyl-4,6-O-benzylidene-3-O-(2-methylnaphthyl)-.beta.-D-glucopyrano-
syl-(1.fwdarw.3)-2-O-benzoyl-4,6-O-benzylidene-.beta.-D-glucopyranosyl-(1.-
fwdarw.3)-4,6-O-benzylidene-2-O-levulinoyl-.beta.-D-glucopyranoside
(Product 7)
[0116] The donor 1 (226 mg, 0.406 mmol) and the acceptor 6 (300 mg,
0.370 mmol) are introduced into anhydrous dichloromethane in a
flask at -80.degree. C., in the presence of a 4 .ANG. molecular
sieve. N-iodosuccinimide (100 mg, 0.444 mmol) and of trimethylsilyl
triflate (6.7 .mu.L, 0.037 mmol) are then added to the medium.
After stirring for 2 hours, the reaction is stopped and the medium
is neutralized with triethylamine, filtered then concentrated.
After purification by silica gel chromatography [toluene/ethyl
acetate (9:1; v/v)], 328 mg of the expected trisaccharide 7 are
obtained with a yield of 68%.
[0117] Product 7: white solid; MP (.degree. C.) 115; Rf (EP/AcOEt,
2:1) 0.5; [.alpha.].sub.D.sup.20 +4.7 (c=1.0,
CH.sub.2Cl.sub.2).
[0118] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 7.95-7.86
(m, 4H, arom. H); 7.70-7.13 (m, 33H, arom. H); 5.51 (s, 1H, H7);
5.43 (s, 1H, H7); 4.94 (d, 1H, H8c, J.sub.H8c-H8'c=12.2 Hz); 4.83
(d, 1H, H8'c); 4.78 (d, 1H, H8a, J.sub.H8a-H8'a=12.4 Hz); 4.50 (s,
1H, H7); 4.49 (d, 1H, H8'a); 2.69-2.47 (m, 2H, H10a); 2.40-2.21 (m,
2H, H11a); 1.95 (s, 3H, H13a) and Table 3a.
[0119] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 206.6
(C12a); 171.1 (C9a); 165.0, 164.8 (2C, OCOPh); 137.3, 137.1, 137.0,
135.4 (quat. arom. C); 133.3, 133.1 (arom. C); 133.0, 132.8 (quat.
arom. C); 129.8, 129.7 (arom. C); 129.4 (quat. arom. C); 129.4,
128.9, 128.7, 128.6, 128.3, 128.2, 128.1, 127.9, 127.8, 127.7,
127.6, 127.5, 126.6, 126.4, 126.1, 126.0, 125.7, 125.6 (arom. C);
102.1 (C7); 101.1 (C7); 100.9 (C7); 73.8 (C8c); 70.4 (C8a); 37.7
(C11a); 29.3 (C13a); 27.6 (C10a) and Table 3b.
[0120] Ultimate analysis (C.sub.76H.sub.72O.sub.20): Theoretical:
C=69.93%, H=5.56%; Measured: C=69.89%, H=5.63%.
[0121] HRMS (ESI.sup.+): [M+Na].sup.+ C.sub.76H.sub.72NaO.sub.20:
theoretical m/z: 1327.4515, measured m/z: 1327.4522; [M+K].sup.+
C.sub.76H.sub.72KO.sub.20: theoretical m/z: 1343.4254, measured
m/z: 1343.4206.
[e]--Preparation of benzyl
2-O-benzoyl-4,6-O-benzylidene-3-O-(2-methylnaphthyl)-.beta.-D-glucopyrano-
syl-(1.fwdarw.3)-2-O-benzoyl-4,6-O-benzylidene-.beta.-D-glucopyranosyl-(1.-
fwdarw.3)-4,6-O-benzylidene-.beta.-D-glucopyranoside (Product
12)
[0122] 7.6 mL of a 1M solution of hydrazine in a pyridine/acetic
acid mixture (3:2, v/v) are added dropwise to a solution of
trisaccharide 7 (500 mg, 0.383 mmol) in pyridine (7.6 mL). After
stirring for 3 hours, the reaction is stopped by the addition of
2.1 mL of 2,4-pentane-dione and the medium is concentrated under
reduced pressure. The residue is then taken up in 50 mL of
dichloromethane and washed with a 10% hydrochloric acid solution, a
saturated aqueous solution of sodium bicarbonate and a saturated
aqueous solution of sodium chloride. After drying (MgSO.sub.4) and
concentration, the product is purified by silica gel chromatography
[petroleum ether/ethyl acetate (2:1; v/v)] in order to produce 377
mg of compound 12 with a yield of 82%.
[0123] Product 12: white solid; MP (.degree. C.) 136; Rf (EP/AcOEt,
2:1) 0.5; [.alpha.].sub.D.sup.20 +13.4 (c=1.0,
CH.sub.2Cl.sub.2).
[0124] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 7.94-7.90
(m, 4H, arom. H); 7.68-7.66 (m, 1H, arom. H); 7.58-7.19 (m, 32H,
arom. 5.54 (s, 1H, H7); 5.52 (s, 1H, H7); 4.95 (d, 1H, H8c,
J.sub.H8c-H8'c=12.4 Hz); 4.88 (d, 1H, H8a, J.sub.H8a-H8'a=11.6 Hz);
4.83 (d, 1H, H8'c); 4.77 (s, 1H, H7); 4.53 (d, 1H, H8'a) and Table
3a.
[0125] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 165.1,
164.7 (2C, OCOPh); 137.3, 137.2, 136.9, 135.3, 133.0, 132.8, 129.2
(8C, quat. arom. C); 133.4, 133.0, 129.8, 129.7, 129.3 (arom. C);
129.2 (quat. arom. C); 129.0, 128.7, 128.5, 128.4, 128.3, 128.2,
128.0, 127.9, 127.8, 127.5, 126.7, 126.4, 126.1, 126.0, 125.7,
125.6 (arom. C); 102.0 (C7); 101.2 (C7); 100.5 (C7); 73.8 (C8c);
71.2 (C8a) and Table 3b.
[0126] Ultimate analysis (C.sub.71H.sub.66O.sub.18): Theoretical:
C=70.64%, H=5.51%; Measured: C=70.49%, H=5.54%.
[0127] HRMS (ESI.sup.+): [M+Na].sup.+ C.sub.71H.sub.66NaO.sub.18:
theoretical m/z: 1229.4147; measured m/z: 1229.4149; [M+K].sup.+
C.sub.7H.sub.66KO.sub.18: theoretical m/z: 1245.3886: measured m/z:
1245.3944; [M-H+2Na].sup.+ C.sub.71H.sub.65Na.sub.2O.sub.18:
theoretical m/z: 1251.3966; measured m/z: 1251.4040.
[f]--Preparation of benzyl
2-O-benzoyl-4,6-O-benzylidene-3-O-(2-methylnaphthyl)-.beta.-D-glucopyrano-
syl-(1.fwdarw.3)-2-O-benzoyl-4,6-O-benzylidene-.beta.-D-glucopyranosyl-(1.-
fwdarw.3)-4,6-O-benzylidene-.beta.-D-glucopyranos-2-uloside
(Product 15a)
benzyl
2-O-benzoyl-4,6-O-benzylidene-3-O-(2-methylnaphthyl)-.beta.-D-gluco-
pyranosyl-(1.fwdarw.3)-2-O-benzoyl-4,6-O-benzylidene-.beta.-D-glucopyranos-
yl-(1.fwdarw.3)-4,6-O-benzylidene-2-C-hydroxyl-.beta.-D-glucopyranoside
(Product 15b)
[0128] 9 mL of a DMSO/acetic acid mixture (2:1, v/v) is added to
the deprotected compound 12 (330 mg, 0.273 mmol). After stirring
for 48 hours at ambient temperature, the medium is diluted with 50
mL of dichloromethane and washed with a saturated aqueous solution
of sodium bicarbonate and with water. After concentration and
several co-evaporations with toluene, the medium is purified by
silica gel chromatography [petroleum ether/ethyl acetate (3:1;
v/v)], in order to produce 180 mg of the oxidized product 15a
(yield=55%) in mixture 2:1 with its hydrated form 15b.
[0129] Product 15a: white solid; Rf (EP/AcOEt, 2:1) 0.3.
[0130] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 7.95-7.91
(m, 4H, arom. H); 7.67-7.60 (m, 1H, arom. H); 7.57-7.10 (m, 32H,
arom. H); 5.53 (s, 1H, H7); 5.41 (s, 1H, H7); 5.40 (dd, 1H, H2c,
J.sub.H1c-H2c=7.8 Hz, J.sub.H2c-H3c=8.4 Hz); 5.32 (d, 1H, H1c);
5.27 (dd, 1H, H2b, J.sub.H1b-H2b=2.4 Hz, J.sub.H2b-H3b=1.6 Hz);
5.19 (d, 1H, H1b); 4.94 (d, 1H, H8c, J.sub.H8c-H8'c=11.7 Hz); 4.82
(d, 1H, H8'c); 4.82 (d, 1H, H8a, J.sub.H8a-H8'a=12.0 Hz); 4.64 (s,
1H, H1a); 4.61 (d, 1H, H8'a); 4.42 (s, 1H, H7); 4.35 (d, 1H, H3a,
J.sub.H3a-H4a=10.4 Hz); 4.35 (t, 1H, H6a, J.sub.H5a-H6a=5.5 Hz);
4.28 (dd, 1H, H6c, J.sub.H5c-H6c=4.9 Hz, J.sub.H6c-H6'c=10.4 Hz);
4.24 (dd, 1H, H4b, J.sub.H3b-H4b=7.5 Hz, J.sub.H4b-H5b=10.6 Hz);
4.11 (dd, 1H, H6b, J.sub.H5b-H6b=4.8 Hz, J.sub.H6b-H6'b=10.2 Hz);
4.01 (dd, 1H, H3b); 3.95 (t, 1H, H3c, J.sub.H3c-H4c=8.4 Hz); 3.91
(t, 1H, H4c, J.sub.H4c-H5c=8.4 Hz); 3.77 (t, 1H, H6'c,
J.sub.H5c-H6'c=10.4 Hz); 3.72 (ddd, 1H, H5b, J.sub.H5b-H6'b=10.2
Hz); 3.63 (ddd, 1H, H5c); 3.56 (t, 1H, H6'a, J.sub.H5a-H6'a=5.5
Hz); 3.55 (dt, 1H, H5a, J.sub.H4a-H5a=9.0 Hz); 3.41 (t, 1H, H6'b);
2.98 (dd, 1H, H4a).
[0131] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 165.2,
165.0 (2C, OCOPh); 137.3, 137.2, 136.8, 135.9, 135.4 (quat. arom.
C); 133.5, 133.3 (arom. C); 133.0, 132.8 (quat. arom. C); 129.8,
129.7, 129.3, 129.2 (arom. C); 129.0 (quat. arom. C); 129.0, 128.9,
128.7, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8,
127.5, 126.7, 126.4, 126.2, 126.1, 126.0, 125.7, 125.6 (arom. C);
101.6 (C7); 101.2 (C7); 100.6 (C7); 98.6 (C1a); 97.6 (C1c); 96.1
(C1b); 81.6 (C4c); 79.9 (C4a); 78.1 (C3c); 77.4 (C4b); 75.9 (C3a);
75.8 (C3b); 73.9 (C8c); 73.1 (C2c); 71.8 (C2b); 70.3 (C8a); 68.9
(C6b); 68.7 (C6c); 68.3 (C6a); 66.4 (C5a); 66.2 (C5c); 64.9
(C5b).
[0132] HRMS (ESI.sup.+): [M+Na].sup.+ C.sub.71H.sub.64NaO.sub.18:
theoretical m/z: 1227.3990; measured m/z: 1227.3988;
[M+Na+CH.sub.3OH].sup.+ C.sub.72H.sub.68NaO.sub.19: theoretical
m/z: 1259.4252, measured m/z: 1259.4245.
[0133] Product 15b:
[0134] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm):
Characteristic signals at 5.24 (d, 1H, H3a, J.sub.H3a-H4a=2.9 Hz);
4.30 (s, 1H, H1a).
[0135] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm):
Characteristic signals at 100.4 (C1a); 93.4 (C2a).
[g]--Preparation of benzyl
2-O-benzoyl-4,6-O-benzylidene-3-O-(2-methylnaphthyl)-.beta.-D-glucopyrano-
syl-(1.fwdarw.3)-2-O-benzoyl-4,6-O-benzylidene-.beta.-D-glucopyranosyl-(1--
43)-4,6-O-benzylidene-.beta.-D-mannopyranoside (Product 18)
[0136] The oxidized compound 15a/15b (180 mg, 0.149 mmol) is
solubilized in a dichloromethane/THF mixture (1:1, v/v) then
L-Selectride (1M in THF; 150 .mu.L, 0.150 mmol) is added at
-78.degree. C. After 45 minutes at -78.degree. C., the reaction is
assumed to be completed and the medium neutralized by the addition
of a few drops of acetic acid. Once the medium has returned to
ambient temperature, 30 mL of dichloromethane are added and the
organic phase is washed with a 10% aqueous hydrochloric acid
solution, a saturated aqueous solution of sodium bicarbonate and
finally with a saturated aqueous solution of sodium chloride. 180
mg of the desired reduced mannoside compound 18 are thus obtained
after drying (MgSO.sub.4) and concentration of the organic
phase.
[0137] Product 18: white solid; MP (.degree. C.) 180; Rf (EP/AcOEt,
2:1) 0.2; [.alpha.].sub.D.sup.20 -4.4 (c=1.0,
CH.sub.2Cl.sub.2).
[0138] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 7.85-7.83
(m, 2H, arom. H); 7.73-7.67 (m, 3H, arom. H); 7.62-7.17 (m, 32H,
arom. H); 5.50 (s, 1H, H7); 5.42 (s, 1H, H7); 5.21 (s, 1H, H7);
4.90 (d, 1H, H8c, J.sub.H8c-H8'c=12.4 Hz); 4.87 (d, 1H, H8a,
J.sub.H8a-H8'a=12.2 Hz); 4.80 (d, 1H, H8'c); 4.59 (d, 1H, H8'a) and
Table 3'a.
[0139] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 164.8,
164.8 (2C, OCOPh); 137.2, 137.1, 136.5, 135.2 (quat. arom. C);
133.2 (arom. C); 132.9, 132.8 (quat. arom. C); 132.8, 129.7, 129.6
(arom. C); 129.4, 129.1 (quat. arom. C); 129.0, 128.9, 128.7,
128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.8, 127.5, 126.7,
126.4, 126.3, 126.1, 126.0, 125.7, 125.6 (arom. C); 101.5 (C7);
101.1 (2C, C7); 73.5 (C8c); 70.4 (C8a) and Table 3'b.
[h]--Preparation of benzyl
4,6-O-benzylidene-3-O-(2-methylnaphthyl)-.beta.-D-glucopyranosyl-(1.fwdar-
w.3)-4,6-O-benzylidene-.beta.-D-glucopyranosyl-(1.fwdarw.3)-4,6-O-benzylid-
ene-.beta.-D-mannopyranoside (Product 19)
[0140] Compound 18 (170 mg, 0.141 mmol) is dissolved in 15 mL of a
methanol/dichloromethane mixture (2:1, v/v) then 2 equivalents of
sodium methylate (0.1M in MeOH, 3 mL, 0.300 mmol) are added. After
stirring for 6 hours at ambient temperature, the medium is
neutralized by the addition of Amberlite IR120-H.sup.+ resin,
filtered then concentrated under reduced pressure. After
chromatography [dichloromethane/methanol (99:1; v/v)], compound 19
is obtained with a yield of 72% (126 mg).
[0141] Product 19: white solid; MP (.degree. C.)>230; Rf
(CH.sub.2Cl.sub.2/MeOH, 97:3) 0.4; [.alpha.].sub.D.sup.20 -43.5
(c=1.0, CH.sub.2Cl.sub.2).
[0142] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 7.70-7.19
(m, 27H, arom. H); 5.47 (s, 1H, H7); 5.44 (s, 1H, H7); 5.42 (s, 1H,
H7); 4.96 (d, 1H, H8c, J.sub.H8c-H8'c=12.0 Hz); 4.88 (d, 1H, H8'c);
4.84 (d, 1H, H8a, J.sub.H8a-H8'a=11.9 Hz); 4.56 (d, 1H, H8'a) and
Table 3'a.
[0143] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 137.1,
137.0, 136.9, 136.2, 135.8, 133.1, 132.8 (7C, quat. arom. C);
129.0, 128.9, 128.8, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0,
127.9, 127.8, 127.6, 127.5, 127.4, 126.8, 126.5, 126.2, 126.1,
126.0, 125.9, 125.8, 125.7 (arom. C); 101.6 (C7); 101.2 (C7); 100.7
(C7); 74.4 (C8c); 70.7 (C8a) and Table 3'b.
[0144] HRMS (ESI.sup.+): [M+Na].sup.+ C.sub.57H.sub.58NaO.sub.16:
theoretical m/z: 1021.3623, measured m/z: 1021.3624; [M+K].sup.+
C.sub.57H.sub.58KO.sub.16: theoretical m/z: 1037.3362, measured
m/z: 1037.3370; [M-H+2Na].sup.+ C.sub.57H.sub.57Na.sub.2O.sub.16:
theoretical m/z: 1043.3442, measured m/z: 1043.3463.
[i]--.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdar-
w.3)-(.alpha.,.beta.)-D-mannopyranose (Product A1)
[0145] The trisaccharide 18 (90 mg, 0.090 mmol), then palladium
acetate (60 mg, 0.267 mmol) are introduced into 10 mL of an ethyl
acetate/methanol/dichloromethane mixture (2:2:1; v/v/v). The medium
is then stirred vigorously at ambient temperature under a hydrogen
atmosphere for 7 days. After filtration on celite, the
hydro-organic phase is extracted with dichloromethane, then brought
to dryness by azeotropic coevaporation with absolute ethanol in
order to lead in quantitative manner to the deprotected compound
(45 mg, 0.090 mmol). 25 mg of the compound formed are then purified
by exclusion chromatography on Sephadex G-10 gel (eluent: water)
and the fractions collected are freeze-dried in order to obtain the
sought product which is presented in the form of a white foam.
[0146] NMR .sup.1H (D.sub.2O, 400 MHz) .delta. (ppm): 5.13 (d, 1H,
H1a.alpha., J.sub.H1a-H2a=1.8 Hz); 4.67 (d, 1H, H1c,
J.sub.H1c-H2c=7.7 Hz); 4.53 (d, 1H, H1b, J.sub.H1b-H2b=8.0 Hz);
4.03 (dd, 1H, H2a, J.sub.H2a-H3a=3.1 Hz); 3.96 (dd, 1H, H3a,
J.sub.H3a-H4a=9.5 Hz); 3.90-3.80 (m, 3H, H6a, H6b, H6c); 3.80-3.73
(m, 1H, H4a); 3.72-3.55 (m, 5H, H5a, H6'a, H3b, H6'b, H6'c);
3.53-3.36 (m, 5H, H2b, H64b, H5b, H3c, H5c); 3.32 (t, 1H, H4c,
J.sub.H3c-H4c=J.sub.H4c-H5c=9.7 Hz); 3.27 (dd, 1H, H2c,
J.sub.H2c-H3c=9.3 Hz).
[0147] NMR .sup.13C (D.sub.2O, 100 MHz) .delta. (ppm): 103.1 (C1c);
100.5 (C1b); 94.1 (C1a.alpha.); 84.5 (C3b); 78.5 (C3a); 76.3 (C5c);
75.9 (2C, C3c, C5b); 75.8 (C2c); 73.1 (C2b); 72.7 (C4a); 69.9
(C4c); 68.6 (C2a); 68.4 (C4b); 65.5 (C5a); 61.2, 61.0 (3C, C6a,
C6b, C6c).
[0148] HRMS (ESI.sup.+): [M+Na].sup.+ C.sub.18H.sub.32NaO.sub.16:
theoretical m/z: 527.1588, measured m/z: 527.1587; [M+K].sup.+
C.sub.18H.sub.32KO.sub.16: theoretical m/z: 543.1327, measured m/z:
543.1343.
Example 2
Synthesis of Different Intermediates
##STR00015##
[0149] 2.1/ benzyl
2-O-benzoyl-4,6-O-benzylidene-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2-O-be-
nzoyl-4,6-O-benzylidene-.beta.-D-glucopyranosyl-(1.fwdarw.3)-4,6-O-benzyli-
dene-2-O-levulinoyl-.beta.-D-glucopyranoside (Product 8)
##STR00016##
[0151] 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (176 mg, 0.774
mmol) is added to a solution of trisaccharide compound 7 (337 mg,
0.258 mmol) in a dichloromethane/methanol mixture (4:1; v/v). After
stirring for 16 hours at ambient temperature, the reaction is
stopped and the medium is diluted with dichloromethane, washed with
a saturated solution of sodium bicarbonate then with water. The
organic phase is dried over MgSO.sub.4, concentrated and the sought
product 8 is obtained after purification on silica gel [petroleum
ether/ethyl acetate (2:1; v/v)] (185 mg, yield=72%).
[0152] Product 8: white solid; Rf (EP/AcOEt, 2:1) 0.2.
[0153] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 8.01-7.95
(m, 2H, arom. H); 7.90-7.84 (m, 2H, arom. H); 7.53-7.18 (m, 24H,
arom. H); 7.15-7.09 (m, 2H, arom. H); 5.39 (s, 1H, H7); 5.37 (s,
1H, H7); 4.74 (d, 1H, H8a, J.sub.H8a-H8'a=12.2 Hz); 4.47 (s, 1H,
H7); 4.45 (d, 1H, H8'a); 2.66 (s, 1H, OH3c); 2.66-2.44 (m, 2H,
H10a); 2.36-2.20 (m, 2H, H11a); 1.91 (s, 311, H13a) and Table
3a.
[0154] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 206.1
(C12a); 171.1 (C9a); 165.7, 164.8 (2C, OCOPh); 137.3, 137.0, 136.9,
133.5, 133.2 (6C, quat. arom. C); 129.9, 129.7, 129.5, 129.4,
129.2, 128.7, 128.4, 128.3, 128.2, 127.9, 127.8, 127.7, 127.6,
126.4, 126.2, 126.0 (arom. C); 102.1, 101.7, 100.4 (3C, C7); 70.4
(C8a); 37.7 (C11a); 29.3 (C13a); 27.7 (C10a) and Table 3b.
2.2/ benzyl
2-O-benzoyl-4,6-O-benzylidene-3-O-(2-methylnaphthyl)-.beta.-D-glucopyrano-
syl-(1.fwdarw.3)-2-O-benzoyl-4,6-O-benzylidene-.beta.-D-glucopyranosyl-(1.-
fwdarw.3)-2-O-benzoyl-4,6-O-benzylidene-.beta.-D-glucopyranosyl-(1.fwdarw.-
3)-4,6-O-benzylidene-2-O-levulinoyl-.beta.-D-glucopyranoside
(Product 9)
##STR00017##
[0156] The donor 1 (26 mg, 0.047 mmol) and the trisaccharide
acceptor 8 (50 mg, 0.043 mmol) are introduced into anhydrous
dichloromethane in a flask at 0.degree. C., in the presence of a 4
.ANG. molecular sieve. After the addition of N-iodosuccinimide
(NIS) (50 mg, 0.051 mmol) and trimethylsilyl triflate (0.65 .mu.L,
0.004 mmol), the mixture is left under vigorous stirring for 30
minutes. Once reaction has stopped, the medium is neutralized with
triethylamine, filtered then concentrated. After purification by
silica gel chromatography [toluene/ethyl acetate (2:1; v/v)], 24 mg
of desired tetrasaccharide 9 are obtained with a yield of 34%.
[0157] Product 9: white solid; MP (.degree. C.) 138; Rf (EP/AcOEt,
2:1) 0.3; [.alpha.].sub.D.sup.20 +8.4 (c=1.0,
CH.sub.2Cl.sub.2).
[0158] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 7.94-7.74
(m, 6H, arom. H); 7.58-7.17 (m, 41H, arom. H); 5.48 (s, 1H, H7);
5.44 (s, 1H, H7); 5.33 (dd, 1H, H2d, J.sub.H1d-H2d=6.9 Hz,
J.sub.H2d-H3d=8.2 Hz); 5.21 (t, 1H, H2c,
J.sub.H1c-H2c=J.sub.H2c-H3c=5.8 Hz); 5.06 (d, 1H, H1c); 4.97 (d,
1H, H1b, J.sub.H1b-H2b=5.8 Hz); 4.96 (d, 1H, H1d); 4.96 (s, 1H,
H7); 4.90 (d, 1H, H8d, J.sub.H8d-H8'd=12.4 Hz); 4.82 (t, 1H, H2b,
J.sub.H2b-H3b=5.8 Hz); 4.80 (d, 1H, H8'd); 4.79 (d, 1H, H8a,
J.sub.H8a-H8'a=12.4 Hz); 4.68 (dd, 1H, H2a, J.sub.H1a-H2a=8.0 Hz,
J.sub.H2a-H3a=8.6 Hz); 4.63 (s, 1H, H7); 4.51 (d, 1H, H8'a); 4.35
(d, 1H, H1a); 4.31 (dd, 1H, H6a, J.sub.H5a-H6a=4.9 Hz,
J.sub.H6a-H6'a=10.6 Hz); 4.21 (dd, 1H, H6d, J.sub.H5d-H6d=4.9 Hz,
J.sub.H6d-H6'd=10.4 Hz); 4.16 (dd, 1H, H6c, J.sub.H5c-H6c=5.6 Hz,
J.sub.H6c-H6'c=10.4 Hz); 4.11 (dd, 1H, H6b, J.sub.H5b-H6b=5.3 Hz,
J.sub.H6b-H6'b=9.1 Hz); 4.08 (dd, 1H, H3c, J.sub.H3c-H4c=8.8 Hz);
4.01 (dd, 1H, H3b, J.sub.H3b-H4b=8.4 Hz); 3.98 (t, 1H, H4c,
J.sub.H4c-H5c=8.8 Hz); 3.90 (dd, 1H, H4d, J.sub.H3d-H4d=8.2 Hz,
J.sub.H4d-H5d=9.1 Hz); 3.90 (dd, 1H, H3a, J.sub.H3a-H4a=9.1 Hz);
3.82 (t, 1H, H3d); 3.73 (t, 1H, H6'd, J.sub.H5d-H6'd=10.4 Hz); 3.67
(t, 1H, H6'a, J.sub.H5a-H6'a=10.6 Hz); 3.62-3.58 (m, 2H, H5c,
H6'c); 3.61 (dd, 1H, H4b, J.sub.H4b-H5b=9.1 Hz); 3.50 (t, 1H, H6'b,
J.sub.H5b-H6'b=9.1 Hz); 3.48 (dt, 1H, H5b); 3.44 (ddd, 1H, H5d);
3.34 (ddd, 1H, H5a, J.sub.H4a-H5a=9.1 Hz); 3.16 (t, 1H, H4a);
2.64-2.50 (m, 2H, H10a); 2.36-2.21 (m, 2H, H11a); 2.00 (s, 3H,
H13a).
[0159] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 206.0
(C12a); 171.1 (C9a); 164.9, 164.8, 164.5 (3C, OCOPh); 137.3, 137.1,
136.9, 135.3 (quat. arom. C); 133.3, 133.2 (arom. C); 133.0, 132.8
(quat. arom. C); 129.7 (arom. C); 129.4 (quat. arom. C); 129.4,
128.9, 128.8, 128.5, 128.4, 128.3, 128.2, 128.0, 127.9, 127.8,
127.7, 127.5, 126.6, 126.4, 126.2, 126.0, 125.9, 125.7, 125.6
(arom. C); 102.0 (C7); 101.1 (C7); 100.8 (2C, C7); 99.7 (C1a); 99.3
(C1d); 97.8 (C1b); 96.8 (C1c); 81.1 (C4d); 78.8 (C4a); 78.5 (C4c);
78.1 (C3d); 77.4 (C4b); 77.2 (C3c); 74.3 (2C, C3a, C3b); 73.6 (2C,
C8d, C2a); 73.4 (C2d); 73.2 (C2b); 72.8 (C2c); 70.5 (C8a); 68.7
(3C, C6b, C6c, C6d); 68.5 (C6a); 66.3 (C5a); 65.9 (C5d); 65.7
(C5c); 65.3 (C5b); 37.7 (C11a); 29.5 (C13a); 27.6 (C10a).
[0160] Ultimate analysis (C.sub.96H.sub.90O.sub.26): Theoretical:
C=69.47%, H=5.47%; Measured: C=69.57%, H=5.58%.
[0161] HRMS (ESI.sup.+): [M+Na].sup.+ C.sub.96H.sub.90NaO.sub.26:
theoretical m/z: 1681.5618, measured m/z: 1681.5619; [M+K].sup.+
C.sub.96H.sub.90KO.sub.26: theoretical m/z: 1697.5357, measured
m/z: 1697.5398.
2.3/ benzyl
4,6-O-benzylidene-3-O-(2-methylnaphthyl)-.beta.-D-glucopyranoside
(Product 10)
##STR00018##
[0163] Sodium (0.7 g, 30.435 mmol) is added to a solution of 3 (5
g, 8.296 mmol) in anhydrous methanol (150 mL). The mixture is
stirred at 50.degree. C. until the starting product is completely
consumed. The medium is then cooled down, neutralized by adding a
stoichiometric quantity of acetic acid and concentrated. The
residue is then dissolved in dichloromethane (250 mL), extracted 3
times with 50 mL of water. The organic phase is dried over
MgSO.sub.4 then concentrated. Purification on silica gel [petroleum
ether/ethyl acetate (3:1; v/v)] makes it possible to obtain 3.7 g
of the debenzoylated compound 10 with a yield of 83%.
[0164] Product 10: white solid; MP (.degree. C.) 148; Rf (EP/AcOEt,
3:1) 0.7; [.alpha.].sub.D.sup.20 -48.1 (c=1.0,
CH.sub.2Cl.sub.2).
[0165] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 7.86-7.80
(m, 2H, arom. H); 7.78-7.72 (m, 1H, arom. H); 7.52-7.30 (m, 14H,
arom. H); 5.60 (s, 1H, H7); 5.10 (d, 1H, H9, J.sub.H9-H9'=12.0 Hz);
4.98 (d, 1H, H9'); 4.94 (d, 1H, H8, J.sub.H8-H8'=11.8 Hz); 4.65 (d,
1H, H8'); 2.46 (s, 1H, OH) and Table 1a.
[0166] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 137.2,
136.8, 135.7, 133.2, 133.0 (5C, quat. arom. C); 129.0, 128.5,
128.3, 128.2, 128.1, 128.0, 127.9, 127.6, 126.8, 126.2, 126.1,
126.0, 125.9, 125.8, 125.4, 125.1 (arom. C); 101.4 (C7); 74.6 (C9);
71.4 (C8) and Table 1b.
[0167] Ultimate analysis (C.sub.31H.sub.30O.sub.6): Theoretical:
C=74.68%, H=6.07%; Measured: C=74.76%, H=6.04%.
[0168] HRMS (ESI.sup.+): [M+Na].sup.+ C.sub.31H.sub.30NaO.sub.6:
theoretical m/z: 521.1940, measured m/z: 521.1948; [M+K].sup.+
C.sub.31H.sub.30KO.sub.6: theoretical m/z: 537.1679, measured m/z:
521.1999.
2.4/ benzyl
2-O-benzoyl-4,6-O-benzylidene-3-O-(2-methylnaphthyl)-.beta.-D-glucopyrano-
syl-(1.fwdarw.3)-4,6-O-benzylidene-.beta.-D-glucopyranoside
(Product 11)
##STR00019##
[0170] 200 .mu.L of a 1M solution of hydrazine in a pyridine/acetic
acid mixture (3:2, v/v) are added dropwise to a solution of the
disaccharide 5 (11 mg, 0.011 mmol) in pyridine (0.2 mL). After
stirring for 1 hour, the reaction is stopped by the addition of 50
.mu.L of 2,4-pentane-dione and the medium is concentrated under
reduced pressure. The residue is then taken up in 20 mL of
dichloromethane and washed with a 10% hydrochloric acid solution, a
saturated aqueous solution of sodium bicarbonate and a saturated
aqueous solution of sodium chloride. After drying the organic phase
on MgSO.sub.4 and evaporation of the solvent, the product is
purified by silica gel chromatography [petroleum ether/ethyl
acetate (2:1; v/v)] in order to produce 6 mg of compound 11 with a
yield of 61%.
[0171] Product 10: white solid; Rf (EP/AcOEt, 2:1) 0.6.
[0172] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 7.93-7.89
(m, 2H, arom. H); 7.70-7.23 (m, 25H, arom. H); 5.54 (s, 1H, H7b);
5.45 (s, 1H, H7a); 4.95 (d, 1H, H8b, J.sub.H8b-H8'b=12.9 Hz); 4.86
(d, 1H, H8'b); 4.85 (d, 1H, H8a, J.sub.H8a-H8'a=11.7 Hz); 4.56 (d,
1H, H8'a) and Tables 2a and 2b.
[0173] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 165.5
(OCOPh); 137.2, 137.1, 136.7, 135.2, 133.1, 132.8 (7C, quat. arom.
C); 133.0, 129.8, 129.7, 129.1, 129.0, 128.5, 128.4, 128.2, 128.0,
127.9, 127.8, 127.6, 126.9, 126.1, 126.0, 125.8, 125.7 (arom. C);
102.0 (C7b); 101.3 (C7a); 74.0 (C8b); 71.3 (C8a) and Table 2c.
[0174] HRMS (ESI.sup.+): [M+Na].sup.+ C.sub.51H.sub.48NaO.sub.12:
theoretical m/z: 875.3044, measured m/z: 875.3040; [M+K].sup.+
C.sub.51H.sub.48KO.sub.12: theoretical m/z: 891.2783, measured m/z:
891.2748
2.5/ benzyl
4,6-O-benzylidene-3-O-(2-methylnaphthyl)-.beta.-D-glucopyranos-2-uloside
(13a) benzyl
4,6-O-benzylidene-2-C-hydroxyl-3-O-(2-methylnaphthyl)-.beta.-D-glucopyran-
oside (13b)
##STR00020##
[0176] Compound 10 (43.5 mg, 0.087 mmol) is put into solution in 1
mL of a DMSO/acetic anhydride mixture (2:1; v/v). After stirring
for 20 hours at ambient temperature, the medium is taken up in 50
mL of ethyl acetate and washed with a saturated aqueous solution of
sodium bicarbonate and a saturated aqueous solution of sodium
chloride. After drying (MgSO.sub.4) and concentration of the
organic phase, the compound is purified by silica gel
chromatography [petroleum ether/ethyl acetate (3:1; v/v)] in order
to obtain 33 mg of a 5:1 mixture of the sought compound 13a and the
hydrated compound 13b, with a yield of 76%.
[0177] White solid; Rf (EP/AcOEt, 3:1) 0.3.
[0178] Product 13a:
[0179] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 7.75-7.66
(m, 3H, arom. H); 7.57-7.54 (m, 1H, arom. H); 7.43-7.20 (m, 13H,
arom. H); 5.49 (s, 1H, H7); 5.03 (d, 1H, H9, J.sub.H9-H9'=12.7 Hz);
4.84 (d, 1H, H8, J.sub.H8-H8'=12.0 Hz); 4.80 (d, 1H, H9'); 4.72 (s,
1H, H1); 4.62 (d, 1H, H8'); 4.35 (dd, 1H, H6, H.sub.H5-H6=4.8 Hz,
J.sub.H5-H6'=10.4 Hz); 4.13 (d, 1H, H3, J.sub.H3-H4=10.2 Hz); 3.90
(t, 1H, H4, J.sub.H4-H5=10.2 Hz); 3.77 (t, 1H, H6',
J.sub.H5-H6'=10.4 Hz); 3.58 (ddd, 1H, H5).
[0180] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 196.7
(C2); 136.8, 135.9, 134.6, 133.1, 133.0 (5C, quat. arom. C); 129.2,
128.6, 128.3, 128.2, 128.1, 127.9, 127.6, 127.1, 126.7, 126.1,
126.0, 125.9, 125.7 (arom. C); 101.2 (C7); 99.0 (C1); 82.0 (C4);
81.8 (C3); 73.1 (C9); 70.5 (C8); 68.5 (C6); 66.4 (C5).
[0181] Product 13b:
[0182] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 7.75-7.66
(m, 3H, arom. H); 7.63-7.59 (m, 1H, arom. H); 7.43-7.20 (m, 13H,
arom. H); 5.49 (s, 1H, H7); 4.97 (d, 1H, H9, J.sub.H9-H9'=11.9 Hz);
4.94 (d, 1H, H9'); 4.84 (d, 1H, H8, J.sub.H8-H8'=11.7 Hz); 4.58 (d,
1H, H8'); 4.38 (s, 1H, H1); 4.28 (dd, 1H, H6, J.sub.H5-H6=4.8 Hz,
J.sub.H6-H6'=10.4 Hz); 3.84 (t, 1H, H4, J.sub.H3-H4=J.sub.H4-H5=9.9
Hz); 3.80 (dd, 1H, H6', J.sub.H5-H6'=9.7 Hz); 3.62 (d, 1H, H3);
3.34 (ddd, 1H, H5); 1.97, 1.95 (2s, 2H, OH2).
[0183] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 136.9,
136.1, 134.8, 133.1, 133.0 (5C, quat. arom. C); 129.0, 128.6,
128.3, 128.2, 128.1, 127.9, 127.6, 127.1, 126.7, 126.2, 126.0,
125.9, 125.7 (arom. C); 101.4 (C7); 100.5 (C1); 93.9 (C2); 79.9
(C4); 79.8 (C3); 74.9 (C9); 71.5 (C8); 68.5 (C6); 66.5 (C5).
2.6/ benzyl
2-O-benzoyl-4,6-O-benzylidene-3-O-(2-methylnaphthyl)-.beta.-D-glucopyrano-
syl-(1.fwdarw.3)-4,6-O-benzylidene-.beta.-D-glucopyranos-2-uloside
(Product 14a)
benzyl
2-O-benzoyl-4,6-O-benzylidene-3-O-(2-methylnaphthyl)-.beta.-D-gluco-
pyranosyl-(1.fwdarw.3)-4,6-O-benzylidene-2-C-hydroxyl-.beta.-D-glucopyrano-
side (Product 14b)
##STR00021##
[0185] Compound 11 (6 mg, 0.007 mmol) is put into solution in 1 mL
of a DMSO/acetic anhydride mixture (2:1; v/v). After stirring for
20 hours at ambient temperature, the medium is taken up in 50 mL of
ethyl acetate and washed with a saturated aqueous solution of
sodium bicarbonate and a saturated aqueous solution of sodium
chloride. After drying (MgSO.sub.4) and concentration of the
organic phase, the compound is purified by silica gel
chromatography [petroleum ether/ethyl acetate (2:1; v/v)] in order
to obtain 6 mg of a 1:5 mixture of the sought compound 14a and the
hydrated compound 14b, with a quantitative yield.
[0186] White solid; Rf (EP/AcOEt, 2:1) 0.3.
[0187] Product 14a:
[0188] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm):
Characteristic signal at 192.2 (C2a).
[0189] HRMS (ESI.sup.+): [M+Na].sup.+ C.sub.51H.sub.46NaO.sub.12:
theoretical m/z: 873.2887; measured m/z: 873.2886; [M'+Na].sup.+
C.sub.51H.sub.48NaO.sub.13: theoretical m/z: 891.2993, measured
m/z: 891.2982.
[0190] Product 14b:
[0191] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 7.92-7.88
(m, 21-1, arom. H); 7.73-7.22 (m, 25H, arom. H); 5.56 (s, 1H, H7);
5.50 (s, 1H, H7); 5.38 (t, 1H, H2b, J.sub.H1b-H2b=J.sub.H2b-H3b=8.0
Hz); 4.96 (d, 1H, H8b, J.sub.H8b-H8'b=12.4 Hz); 4.90 (d, 1H, H8a,
J.sub.H8a-H8'a=12.0 Hz); 4.89 (d, 1H, H8'b); 4.86 (d, 1H, H1b);
4.58 (d, 1H, H8'a); 4.34 (dd, 1H, H6b, J.sub.H5b-H6b=4.9 Hz,
J.sub.H6b-H6'b=10.6 Hz); 4.30 (s, 1H, H1a); 4.27 (dd, 1H, H6a,
J.sub.H5a-H6a=5.1 Hz, J.sub.H6a-H6'a=10.6 Hz); 3.99-3.76 (m, 5H,
H4a, H6'a, H3b, H4b, H6'b); 3.75 (d, 1H, H3a, J.sub.H3a-H4a=9.5
Hz); 3.48 (dt, 1H, H5b, J.sub.H4b-H5b=J.sub.H5b-H6'b=9.5 Hz); 3.36
(dt, 1H, H5a, J.sub.H4a-H5a=J.sub.H5a-H6'a=10.0 Hz).
[0192] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 166.3
(OCOPh); 137.2, 137.1, 136.1, 135.1, 132.9 (quat. arom. C); 133.3,
133.0, 129.9, 129.5, 129.1, 129.0, 128.6, 128.5, 128.4, 128.3,
128.2, 128.1, 128.0, 127.9, 127.6, 127.0, 126.3, 126.2, 126.1,
125.9, 125.8 (arom. C); 103.0 (C1b); 101.2, 101.0 (C7); 100.4
(C1a); 93.4 (C2a); 81.9 (C3a); 81.0 (C4b); 78.1 (C4a); 77.6 (C3b);
74.4 (C2b); 73.9 (C8b); 71.4 (C8a); 68.6 (C6a); 68.4 (C6b); 66.8
(C5a); 65.9 (C5b).
2.7/ benzyl
4,6-O-benzylidene-3-O-(2-methylnaphthyl)-(1-D-mannopyranoside
(Product 16)
##STR00022##
[0194] An equivalent of L-Selectride (1M in THF; 70 .mu.L, 0.070
mmol) is added to a solution of the 13a/13b mixture (33 mg, 0.067
mmol) in THF (0.5 mL) at -80.degree. C. After stirring for 15
minutes at -80.degree. C., the medium is diluted with 10 mL of
dichloromethane and washed with a 10% aqueous hydrochloric acid
solution, a saturated aqueous solution of sodium bicarbonate and a
saturated aqueous solution of sodium chloride. After drying over
MgSO.sub.4 and evaporation of the solvent, the compound is purified
by silica gel chromatography [petroleum ether/ethyl acetate (3:1;
v/v)] in order to obtain 28 mg of the sought mannoside compound 16
with a yield of 85%.
[0195] Product 16: white solid; Rf (EP/AcOEt, 3:1) 0.3.
[0196] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 7.85-7.79
(m, 3H, arom. H); 7.73-7.71 (m, 1H, arom. H); 7.55-7.30 (m, 13H,
arom. H); 5.65 (s, 1H, H7); 4.99 (d, 1H, H9, J.sub.H9-H9'=12.7 Hz);
4.96 (d, 1H, H9'); 4.94 (d, 1H, H8, J.sub.H8-H8'=12.0 Hz); 4.65 (d,
1H, H8'); 4.52 (d, 1H, H1, J.sub.H1-H2=1.0 Hz); 4.37 (dd, 1H, H6,
J.sub.H5-H6=5.1 Hz, J.sub.H6-H6'=10.4 Hz); 4.21 (t, 1H, H4,
J.sub.H3-H4=J.sub.H4-H5=9.4 Hz); 4.14 (dd, 1H, H2, J.sub.H2-H3=3.3
Hz); 3.94 (t, 1H, H6', J.sub.H5-H6'=10.4 Hz); 3.66 (dd, 1H, H3);
3.33 (ddd, 1H, H5).
[0197] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 137.4,
136.5, 135.3, 133.1, 133.0 (5C, quat. arom. C); 129.2, 129.0,
128.6, 128.5, 128.3, 128.2, 128.1, 127.9, 127.6, 126.7, 126.2,
126.1, 125.9, 125.7 (arom. C); 101.6 (C7); 98.6 (C1;
.sup.1J.sub.C1-H1=158 Hz); 78.4 (C4); 76.4 (C3); 72.4 (C9); 70.7
(C8); 70.0 (C2); 68.6 (C6); 66.9 (C5).
2.8/ benzyl
2:0-benzoyl-4,6-O-benzylidene-3-O-(2-methylnaphthyl)-.beta.-D-glucopyrano-
syl-(1.fwdarw.3)-4,6-O-benzylidene-.beta.-D-mannopyranoside
(Product 17)
##STR00023##
[0199] L-Selectride (1M solution in THF; 6 .mu.L, 0.006 mmol) is
added to the compound 14a/14b (5 mg, 0.006 mmol) in solution in 1
mL of THF at -90.degree. C. After stirring for 1 hour at low
temperature, the medium is neutralized with acetic acid, diluted
with 20 mL of dichloromethane and washed with a 10% aqueous
hydrochloric acid solution, a saturated solution of sodium
bicarbonate and a saturated aqueous solution of sodium chloride.
After drying (MgSO.sub.4) and concentration of the organic phase, 5
mg of the sought compound 17 (0.006 mmol) are obtained with a
quantitative yield.
[0200] Product 17: white solid; Rf (EP/AcOEt, 2:1) 0.3.
[0201] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 7.90-7.20
(m, 20H, arom. H); 5.55 (s, 1H, H7); 5.48 (s, 1H, H7); 5.35 (t, 1H,
H2b, J.sub.H1b-H2b=J.sub.H2b-H3b=7.5 Hz); 4.94 (d, 1H, H8b,
J.sub.H8b-H8'b=12.2 Hz); 4.86 (d, 1H, H8'b); 4.81 (d, 1H, H8a,
J.sub.H8a-H8'a=11.9 Hz); 4.79 (d, 1H, H1b); 4.56 (d, 1H, H8'a);
4.37 (d, 1H, H1a, J.sub.H1a-H2=0.9 Hz); 4.35-4.17 (m, 2H, H6a,
H6b); 4.07 (t, 1H, H4b, J.sub.H3b-H4b=J.sub.H4b-H5b=9.3 Hz); 3.94
(t, 1H, H3a, J.sub.H2a-H3a=J.sub.H3a-H4a=9.0 Hz); 3.90-3.68 (m, 5H,
H2a, H4a, H3b, H6'a, H6'b); 3.56-3.24 (m, 2H, H5a, H5b).
[0202] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 166.3
(OCOPh); 137.2, 135.1, 133.2 (quat. arom. C); 129.9, 129.8, 129.7,
129.0, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8,
127.5, 127.0, 126.3, 126.1, 126.0, 125.9, 125.8, 125.7 (arom. C);
101.2, 101.0 (C7); 100.4 (C1b); 98.3 (C1a); 81.8 (C3a); 80.9 (C4b);
78.4 (C4a); 77.7 (C3b); 74.0 (C2b); 73.7 (C8b); 70.5 (C8a); 69.4
(C2a); 68.6, 68.5 (C6a, C6b); 67.0 (C5a); 66.3 (C5b).
Example 3
Preparation of
.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-mannopyranose
(Product A2)
##STR00024##
[0204] The desired product was prepared according to a protocol
similar to that described for the product A1 by applying the
successive stages [d], [c], [d], [c], then [e] to [i] to the
product 9.
[0205] In this way the product A2 which is presented in the form of
a white foam was obtained; Rf (AcOEt/iPrOH/H.sub.2O, 3:2:2)
0.2.
[0206] NMR .sup.1H (D.sub.2O, 400 MHz) .delta. (ppm): 5.19 (d, 1H,
H1a.alpha., J.sub.H1a-H2a=2.2 Hz).
[0207] Other Signals:
[0208] NMR .sup.1H (D.sub.2O, 400 MHz) .delta. (ppm): 4.75 (d, 3H,
3H1, J.sub.H1-H2=8.0 Hz); 4.71 (d, 2H, 2H1, J.sub.H1-H2=7.8 Hz);
3.93-3.81 (m, 8H, H3a, H4a, H6a, H6b, H6c, H6d, H6e, H6f);
3.80-3.63 (m, 11H, H2a, H3b, H3c, H3d, H3e, H6'a, H6'b, H6'c, H6'd,
H6'e, H6'f); 3.55-3.40 (m, 11H, H2b, H2c, H2d, H2e, H3f, H4b, H4c,
H4d, H4e, H5a, H5b, H5c, H5d, H5e, H5f); 3.36 (t, 1H, H4f,
J.sub.H3f-H4f=J.sub.H4f-H5f=9.3 Hz); 3.31 (dd, 1H, H2f,
J.sub.H1f-H2f=8.0 Hz, J.sub.H2f-H3f=9.3 Hz).
[0209] HRMS (ESI.sup.+): [M+Na].sup.+ C.sub.36H.sub.62NaO.sub.31:
theoretical m/z: 1013.3173, measured m/z: 1013.3172; [M+K].sup.+
C.sub.36H.sub.62KO.sub.31: theoretical m/z: 1029.2912, measured
m/z: 1029.2920.
Example 4
Preparation of
.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-mannitol (product B1) according to reaction diagram 1
##STR00025##
[0211] The deprotected trisaccharide A1 (25 mg, 0.049 mmol) is
introduced into 10 mL of a methanol/water mixture (4:1; v/v).
Sodium borohydride (19 mg, mmol) is added at ambient temperature
and the medium is left under stirring for 10 days. The medium is
then neutralized by the addition of a few drops of acetic acid then
brought to dryness by coevaporation with a methanol/acetic acid
mixture (9:1, v/v), then by coevaporation with methanol. After
freeze-drying, the compound is purified by Sephadex gel G-10
permeation chromatography (eluent: water) and the fractions
collected are freeze-dried in order to obtain the sought product B1
which is presented in the form of a white foam.
[0212] NMR .sup.1H (D.sub.2O, 400 MHz) .delta. (ppm): 4.82 (d, 1H,
Hie, J.sub.H1c-H2c=8.0 Hz); 4.65 (d, 1H, H1b, J.sub.H1b-H2b=8.1
Hz); 4.09-3.90 (m, 7H, H1a, H2a, H3a, H5a, H6a, H6b, H6c);
3.86-3.66 (m, 6H, H1'a, H4a, H6'a, H3b, H6'b, H6'c); 3.64-3.38 (m,
7H, H2b, H4b, H5b, H2c, H3c, H4c, H5c).
[0213] NMR .sup.13C (D.sub.2O, 100 MHz) .delta. (ppm): 103.1 (C1c);
102.5 (C1b); 84.4 (C3b); 77.3 (C3a); 76.3 (C5c); 75.8 (C3c); 75.5
(C5b); 73.7 (C2c); 73.4 (C2b); 70.9, 70.8 (2C, C2a, C5a); 69.9
(C4c); 69.6 (C4a); 68.8 (C4b); 63.4 (C6a); 62.6 (C1a); 61.2, 61.0
(2C, C6b, C6c).
Example 5
Preparation of the Product A1 According to Reaction Diagram 2
##STR00026##
[0214] 5.1/ Preparation of benzyl
2,3,4,6-tetra-O-acetyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-a-
cetyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-4,6-O-benzylidene-2-O-levulinoy-
l-.beta.-D-glucopyranoside (Product 21)
[0215] The donor 20 [Blattner, R.; Furneaux, R. H.; Pakuiski, Z.
Carbohydr. Res. 2006, 341, 2115-2125.] (50 mg, 0.064 mmol) and the
acceptor 4 (27 mg, 0.058 mmol) are introduced into anhydrous
dichloromethane in a flask at -50.degree. C., in the presence of a
4 .ANG. molecular sieve. Trimethylsilyl triflate (0.53 .mu.L, mmol)
is then added and the mixture is stirred vigorously for 2 hours.
Once the reaction has stopped, the medium is neutralized with
triethylamine, filtered then concentrated. After purification by
silica gel chromatography [petroleum ether/ethyl acetate (2:1;
v/v)], 22 mg of the expected trisaccharide 21 are obtained with a
yield of 35%.
[0216] Product 21: amorphous white solid; Rf (EP/AcOEt, 1:1)
0.2.
[0217] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 7.46-7.43
(m, 2H, arom. H); 7.37-7.27 (m, 8H, arom. H); 5.53 (s, 1H, H7a);
5.10 (t, 1H, H3c, J.sub.H2c-H3c=J.sub.H3c-H4c=9.3 Hz); 5.04 (t, 1H,
H4c, J.sub.H4c-H5c=9.3 Hz); 5.02 (t, 1H, H2a, J.sub.H1a-H2a=7.7 Hz,
J.sub.H2a-H3a=9.5 Hz); 4.95 (dd, 1H, H2b, J.sub.H1b-H2b=8.2 Hz,
J.sub.H2b-H3b=6.0 Hz); 4.93 (dd, 1H, H4b, J.sub.H3b-H4b=7.3 Hz,
J.sub.H4b-H5b=9.7 Hz); 4.87 (dd, 1H, H2c, J.sub.H1c-H2c=8.2 Hz);
4.86 (d, 1H, H8a, J.sub.H8a-H8'a=12.2 Hz); 4.62 (d, 1H, H1c); 4.57
(d, 1H, H8'a); 4.55 (d, 1H, H1b); 4.50 (d, 1H, H1a); 4.34 (dd, 1H,
H6c, J.sub.H5c-H6c=2.0 Hz, J.sub.H6c-H6'c=12.4 Hz); 4.33 (dd, 1H,
H6a, J.sub.H5a-H6a=4.6 Hz, J.sub.H6a-H6'a=10.4 Hz); 4.15-4.01 (m,
3H, H6b, H6'b, H6'c); 3.93 (t, 1H, H3a, J.sub.H3a-H4a=9.5 Hz); 3.91
(dd, 1H, H3b); 3.79 (t, 1H, H6'a, J.sub.H5a-H6'a=10.4 Hz); 3.69 (t,
1H, H4a, J.sub.H4a-H5a=9.5 Hz); 3.69 (ddd, 1H, H5c,
J.sub.H5c-H6'c=4.0 Hz); 3.54 (ddd, 1H, H5b, J.sub.H5b-H6b=2.4 Hz,
J.sub.H5b-H6'b=4.0 Hz); 3.40 (ddd, 1H, H5a); 2.87-2.80 (m, 1H,
H10a); 2.70-2.62 (m, 2H, H10'a, H11a); 2.50-2.42 (m, 1H, H11'a);
2.20 (s, 3H, H13a); 2.04, 1.99, 1.97, 1.96, 1.94 (6s, 21H,
OCOCH.sub.3).
[0218] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 206.1
(C12a); 171.0 (C9a); 170.8, 170.5, 170.4, 169.3, 169.2, 169.1 (7C,
OCOCH.sub.3); 137.0, 136.8 (2C, quat. arom. C); 129.0, 128.5,
128.4, 128.3, 128.1, 128.0, 127.9, 127.8, 125.9 (arom. C); 100.9
(C7a); 100.7 (C1c); 100.1 (C1b); 99.8 (C1a); 78.7 (C3b); 78.6
(C4a); 77.7 (C3a); 73.8 (C2a); 73.0 (C3c); 72.6 (C2b); 71.5 (C5b);
71.4 (C5c); 70.9 (C2c); 70.7 (C8a); 68.5 (C6a); 68.1 (C4c); 68.0
(C4b); 66.5 (C5a); 62.2 (C6b); 61.6 (C6c); 37.6 (C11a); 30.1
(C13a); 27.6 (C10a); 21.0, 20.9, 20.7, 20.6, 20.5, 20.4 (7C,
OCOCH.sub.3).
5.2/ Preparation of benzyl
2,3,4,6-tetra-O-acetyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-a-
cetyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2-O-benzoyl-4,6-O-benzylidene-.-
beta.-D-glucopyranosyl-(1.fwdarw.3)-4,6-O-benzylidene-2-O-levulinoyl-.beta-
.-D-glucopyranoside (Product 22)
[0219] The disaccharide donor 37 (106 mg, 0.136 mmol) and the
acceptor 71 (100 mg, 0.123 mmol) are introduced into anhydrous
dichloromethane in a flask at -50.degree. C., in the presence of a
4 .ANG. molecular sieve. Trimethylsilyl triflate (1.1 .mu.L, 0.006
mmol) is added and the reaction medium is stirred for 60 minutes.
The medium is neutralized with triethylamine, filtered then
concentrated. After purification by silica gel chromatography
[petroleum ether/ethyl acetate (1:1; v/v)], 146 mg of the
tetrasaccharide orthoester are obtained with a yield of 83%. The
compound thus obtained is directly involved in an intramolecular
rearrangement reaction in the presence of trimethylsilyl triflate
(2.5 .mu.L, 0.014 mmol) in dichloromethane at 0.degree. C. After
stirring for 3 hours, the medium is neutralized with triethylamine,
then concentrated. After purification by silica gel chromatography
[petroleum ether/ethyl acetate (1:1; v/v)], 101 mg of the expected
product 22 are obtained with a yield of 69%.
[0220] Product 22: white solid; MP (.degree. C.) 119; Rf (EP/AcOEt,
1:1) 0.3.
[0221] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 8.02-7.99
(m, 2H, arom. H); 7.61-7.12 (m, 18H, arom. H); 5.59 (s, 1H, H7b);
4.83 (d, 1H, H8a, J.sub.H8a-H8'a=12.2 Hz); 4.57 (s, 1H, H7a); 4.56
(d, 1H, H8'a); 2.73-2.65 (m, 1H, H10a); 2.63-2.51 (m, 1H, H10'a);
2.48-2.40 (m, 1H, H11a); 2.35-2.26 (m, 1H, H11b); 2.08 (s, 3H,
H13a); 2.08, 2.04, 2.01, 2.00, 1.97, 1.94 (7s, 21H, OCOCH.sub.3)
and Table 4a.
[0222] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 206.0
(C12a); 171.2 (C9a); 170.8, 170.5, 170.3, 169.4, 169.3, 169.1,
168.7 (6C, OCOCH.sub.3); 165.0 (OCOPh); 137.3, 136.9, 136.7 (3C,
quat. arom. C); 133.3, 129.8, 129.7 (arom. C); 129.6 (quat. arom.
C); 129.5, 128.5, 128.4, 128.3, 128.2, 127.9, 127.8, 126.5, 126.0,
125.8 (arom. C); 102.2 (C7b); 99.7 (C7a); 70.7 (C8a); 37.6 (C11a);
29.4 (C13a); 27.7 (C10a); 21.0, 20.9, 20.6, 20.5, 20.4, 20.3 (7C,
OCOCH.sub.3) and Table 4b.
[0223] HRMS (ESI.sup.+): [M+Na].sup.+ C.sub.71H.sub.80NaO.sub.31:
theoretical m/z: 1451.4581, measured m/z: 1451.4579; [M+K].sup.+
C.sub.71H.sub.80KO.sub.31: theoretical m/z: 1467.4321, measured
m/z: 1467.4346.
5.3/ benzyl
2,3,4,6-tetra-O-acetyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-a-
cetyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2-O-benzoyl-4,6-O-benzylidene-.-
beta.-D-glucopyranosyl-(1.fwdarw.3)-4,6-O-benzylidene-.beta.-D-glucopyrano-
side (Product 23)
[0224] The tetrasaccharide 22 (430 mg, 0.301 mmol) is put into
solution in 6 mL of pyridine then 6 mL of a 1M solution of
hydrazine in a pyridine/acetic acid mixture (3:2, v/v) is added
dropwise. After stirring for 3 hours, the reaction is stopped by
the addition of 1.7 mL of 2,4-pentane-dione and concentrated under
reduced pressure. The medium is taken up in 50 mL of
dichloromethane and washed with a 10% hydrochloric acid solution, a
saturated aqueous solution of sodium bicarbonate and a saturated
aqueous solution of sodium chloride. After drying (MgSO.sub.4) and
concentration, the product is purified by silica gel chromatography
[petroleum ether/ethyl acetate (1:1; v/v)] in order to produce 260
mg of the deprotected compound 23 with a yield of 65%.
[0225] Product 23: white solid; MP (.degree. C.) 136; Rf (EP/AcOEt,
1:1) 0.3; [.alpha.].sub.D.sup.20 -29.6 (c=1.0,
CH.sub.2Cl.sub.2).
[0226] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 8.11-8.09
(dd, 2H, arom. H); 7.67-7.17 (m, 18H, arom. H); 5.61 (s, 1H, H7);
5.08 (s, 1H, 117); 4.92 (d, 1H, H8a, J.sub.H8a-H8'a=11.7 Hz); 4.61
(d, 1H, H8'a); 2.09, 2.03, 2.01, 2.00, 1.99 (7s, 21H, OCOCH.sub.3)
and Table 4a.
[0227] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 170.8,
170.5, 170.2, 169.3, 169.2, 169.0, 168.6 (7C, OCOCH.sub.3); 165.1
(OCOPh); 137.1, 137.0, 136.5 (3C, quat. arom. C); 133.3 (arom. C);
129.5 (quat. arom. C); 129.7, 129.1, 128.9, 128.8, 128.7, 128.5,
128.4, 128.2, 128.1, 128.0, 127.9, 127.8, 126.0, 125.8, 125.2
(arom. C); 101.4 (C7); 100.4 (C7); 71.3 (C8a); 20.6, 20.5, 20.4,
20.3, 20.2 (7C, OCOCH.sub.3) and Table 4b.
5.4/ Preparation of benzyl
2,3,4,6-tetra-O-acetyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-a-
cetyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2-O-benzoyl-4,6-O-benzylidene-.-
beta.-D-glucopyranosyl-(1.fwdarw.3)-4,6-O-benzylidene-.beta.-D-glucopyrano-
s-2-uloside (Product 24a) and of benzyl
2,3,4,6-tetra-O-acetyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-a-
cetyl-fi-D-glucopyranosyl-(1.fwdarw.3)-2-O-benzoyl-4,6-O-benzylidene-.beta-
.-D-glucopyranosyl-(1.fwdarw.3)-4,6-O-benzylidene-2-C-hydroxyl-.beta.-D-gl-
ucopyranoside (Product 24b)
[0228] Pyridine (0.5 mL) and Dess-Martin periodinane (2.6 mL of a
15% solution) are added to a solution of the product 23 (260 mg,
0.195 mmol) in 5 mL of anhydrous dichloromethane. After 24 hours at
ambient temperature, ethyl ether is added to the medium. The
resultant precipitate is filtered on celite and the filtrate
concentrated under reduced pressure. The residue is finally
purified by silica gel chromatography [petroleum ether/ethyl
acetate (1:1; v/v)], in order to produce 208 mg of the oxidized
product 24a (yield=80%) in mixture (1:1) with its hydrated form
24b.
[0229] 24a/24b: white solid; Rf (EP/AcOEt, 1:1) 0.2.
[0230] Product 24a: NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta.
(ppm): Characteristic signals 5.25 (s, 1H, H1a).
[0231] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm):
Characteristic signals 196.6 (C2a); 96.8 (C1a); 70.6 (C8a).
[0232] Product 24b: NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta.
(ppm): Characteristic signals 4.87 (s, 1H, H1a).
[0233] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm):
Characteristic signals 98.8 (C1a); 93.7 (C2a); 71.4 (C8a).
[0234] Other Signals (24a/24b):
[0235] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 8.08-8.03
(m, 2H, arom. H); 7.67-7.45 (m, 8H, arom. H); 7.40-7.10 (m, 30H,
arom. H); 5.65 (s, 1H, H7); 5.59 (s, 1H, H7); 5.26-5.18 (m, 2H,
2H2b); 5.14-4.80 (m, 14H, H2c, 2H2d, 2H3d, 2H4a, 2H4b, 2H8a, 2H1);
4.73 (d, 1H, H8a, J.sub.H8a-H8'a=11.9 Hz); 4.66-4.48 (m, 6H, H3a,
H8'a, 2H1, H6, H7); 4.42-4.26 (m, 61-1, H3a, H1, H7, 3H6);
4.20-3.40 (m, 28H, 2H3b, 2H3c, 2H4c, 2H4d, 2H5a, 2H5b, 2H5c, 2H5d,
2H6'a, 2H6'b, 2H6'c, 2H6'd, 4H6); 2.08, 2.06, 2.04, 2.01, 2.00,
1.99, 1.98, 1.97, 1.96, 1.95 (14s, 42H, OCOCH.sub.3).
[0236] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 170.8,
170.5, 170.3, 169.4, 169.3, 169.2, 169.1, 168.8 (10C, OCOCH.sub.3);
165.3 (2C, OCOPh); 137.1, 137.0, 136.6, 136.0, 135.6 (6C, quat.
arom. C); 133.7, 133.6, 129.9, 129.4, 129.3, 129.2, 129.1, 128.8,
128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 127.9, 127.7, 126.4,
126.2, 125.9, 125.8 (arom. C, 2 quat. arom. C); 101.8, 101.6,
100.4, 100.2 (4C, C7); 100.9, 100.8, 100.6, 99.2, 98.8, 96.8 (8C,
C1); 80.6, 79.8, 79.3, 79.0, 78.7, 78.1, 77.8, 77.4, 77.2, 77.1
(10C, C3a, C3b, C3c, C4a, C4b); 74.8 (2C, C2b); 73.3, 73.0, 72.8,
72.3 (4C, C2c, C3d); 71.6, 71.5 (4C, C5c, C5d); 71.0, 70.8 (2C,
C2d); 68.7, 68.6, 68.4 (4C, C6a, C6b); 68.0, 67.9 (4C, C4c, C4d);
67.0, 66.8 (2C, C5a); 65.6, 65.1 (2C, C5b); 62.0 (2C, C6d); 61.6
(2C, C6c); 21.1, 20.7, 20.6, 20.5, 20.4, 20.3 (14C,
OCOCH.sub.3).
5.5/ benzyl
2,3,4,6-tetra-O-acetyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-a-
cetyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2-O-benzoyl-4,6-O-benzylidene-.-
beta.-D-glucopyranosyl-(1.fwdarw.3)-4,6-O-benzylidene-.beta.-D-mannopyrano-
side (Product 25)
[0237] L-Selectride (1M solution in THF; 150 .mu.L, 0.150 mmol) is
added to the product 24a/24b (66 mg, 0.050 mmol) in solution in a
dichloromethane/THF mixture at -90.degree. C. After stirring for 2
hours at low temperature, the medium is neutralized by the addition
of acetic acid, diluted with 20 mL of dichloromethane and washed
with a saturated aqueous solution of hydrochloric acid, a saturated
aqueous solution of sodium bicarbonate and water. After drying over
MgSO.sub.4 and concentration, the medium is purified by silica gel
chromatography [dichloromethane/methanol (98:2; v/v)], in order to
produce the desired reduced product 25 (66 mg, 0.050 mmol) with a
quantitative yield.
[0238] Product 25: white solid; Rf (CH.sub.2Cl.sub.2/MeOH, 98:2)
0.2.
[0239] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 8.08-8.03
(m, 2H, arom. H); 7.67-7.10 (m, 18H, arom. H); 5.62 (s, 1H, H7);
5.22 (m, 1H, H2b, J.sub.H1b-H2b=J.sub.H2b-H3b=5.1 Hz); 5.04 (t, 1H,
H3d, J.sub.H2d-H3d=J.sub.H3d-H4d=8.0 Hz); 5.03-4.91 (m, 5H, H1b,
H2c, H4c, H4d, H7); 4.90 (d, 1H, H8a, J.sub.H8a-H8'a=11.9 Hz); 4.84
(dd, 1H, H2d, J.sub.H1d-H2d=8.2 Hz); 4.67 (d, 1H, H1c,
J.sub.H1c-H2c=8.0 Hz); 4.63 (d, 1H, H8'a); 4.47 (d, 1H, H1a,
J.sub.H1a-H2a<1.0 Hz); 4.44 (d, 1H, Hid); 4.39-4.31 (m, 2H, H6a,
H6c); 4.18-3.94 (m, 8H, H2a, H3b, H4a, H4b, H6b, H6d, H6'c, H6'd);
3.93-3.84 (m, 2H, H3a, H6'a); 3.75 (t, 1H, H3c,
J.sub.H2c-H3c=J.sub.H3c-H4c=9.3 Hz); 3.64 (t, 1H, H6'b,
J.sub.H5b-H6'b=J.sub.H6b-H6'b=10.0 Hz); 3.62-3.52 (m, 3H, H5b, H5c,
H5d); 3.34 (dt, 1H, H5a, J.sub.H4a-H5a=J.sub.H5a-H6'a=9.8 Hz,
J.sub.H5a-H6a=4.6 Hz); 2.05, 2.00, 1.97, 1.95, 1.94 (7s, 21H,
OCOCH.sub.3).
[0240] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 170.8,
170.4, 170.3, 169.3, 169.2, 169.1, 168.9 (7C, OCOCH.sub.3); 165.0
(OCOPh); 137.2, 137.1, 136.4, 129.2 (4C, quat. arom. C); 133.6,
129.7, 129.0, 128.8, 128.6, 128.5, 128.4, 128.3, 128.2, 128.0,
127.8, 126.1, 125.8 (arom. C); 101.7, (C7); 100.7 (C1d); 100.5
(C7); 99.7 (C1c); 98.3 (C1a); 97.3 (C1b); 78.7 (C3c); 78.4 (C3b);
77.5 (C4b); 76.3 (C4a); 75.7 (C3a); 74.2 (C2b); 72.9 (C3d); 72.4
(C2c); 71.5 (2C, C5c, C5d); 70.8 (C2d); 70.6 (C7a); 68.7 (C2a);
68.6 (C6b); 68.5 (C6a); 68.1 (C4c); 67.9 (C4d); 67.2 (C5a); 65.9
(C5b); 61.9 (C6d); 61.5 (C6c); 20.7, 20.6, 20.5, 20.4 (7C,
OCOCH.sub.3).
5.6/ Preparation of the Product A1
[0241] Compound 25 (60 mg, 0.045 mmol) is dissolved in a
dichloromethane/methanol mixture (2:1, v/v; 10 mL) then 2
equivalents of sodium methylate (0.1M in MeOH, 0.9 mL, 0.090 mmol)
are added. After stirring for 6 hours at ambient temperature, the
medium is neutralized by the addition of Amberlite IR120-H.sup.+
resin, filtered then concentrated under reduced pressure. The
residue thus obtained is then dissolved in 10 mL of an ethyl
acetate/methanol/dichloromethane mixture (2:2:1; v/v/v). After the
addition of palladium acetate (60 mg, 0.267 mmol), the medium is
stirred vigorously at ambient temperature under a hydrogen
atmosphere for 7 days. After filtration on celite, the
hydro-organic phase is extracted with dichloromethane, then brought
to dryness by azeotropic coevaporation with absolute ethanol. The
product A1 (30 mg) is then isolated with a quantitative yield in
the last three stages.
Example 6
##STR00027## ##STR00028##
[0243] The product 1a is obtained as described in the Patent
Application FR2804684
6.1/ Preparation of
2,3,4,6-tetra-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O--
benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D--
glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D-glucopyranosyl-(1-
.fwdarw.3)-1,2,4,6-tetra-O-benzoyl-(.alpha.,.beta.)-D-glucopyranose
(Product 2a)
[0244] The oligosaccharide 1a (150 mg, 0.181 mmol) is dissolved in
pyridine (15 mL). The reaction medium is then cooled down to
0.degree. C. then benzoyl chloride (4.2 mL, 36 mmol) is added.
After stirring for 2 days at ambient temperature, the medium is
evaporated, dissolved in dichloromethane then washed with a 10%
aqueous hydrochloric acid solution, a saturated solution of sodium
bicarbonate and a saturated solution of sodium chloride until
neutrality is reached. The organic phase is then dried over
MgSO.sub.4 and evaporated. The residue is purified by silica gel
chromatography [petroleum ether/ethyl acetate (3:2; v/v)] in order
to produce the perbenzoylated derivative 2a in the form of a
mixture of anomers .alpha./.beta.:1/1 with a yield of 94% (444 mg,
0.170 mmol).
[0245] Product 2a: colourless oil; Rf (EP/AcOEt, 1:1) 0.4.
[0246] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm):
Characteristic signals for 2a 6.65 (d, 1H, H1a, J.sub.H1a-H2a=3.6
Hz); 5.47 (t, 1H, H4a, J.sub.H3a-H4a=J.sub.H4a-H5a=9.4 Hz); 4.64
(t, 1H, H3a, J.sub.H2a-H3a=9.4 Hz). Characteristic signals for
2a.quadrature..quadrature.6.07 (d, 1H, H1a, J.sub.H1a-H2a=7.1 Hz);
5.57 (t, 1H, H4a, J.sub.H3a-H4a=J.sub.H4a-H5a=8.9 Hz); 4.25 (dt,
1H, H5a, J.sub.H5a-H6a=4.3 Hz, J.sub.H5a-H6'a=8.9 Hz).
[0247] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm):
Characteristic signals for 2a 87.7 (C1a); 76.3 (C3a); 72.4 (C2a);
70.3 (C5a); 67.6 (C4a). Characteristic signals for 2a 89.7 (C1a);
78.4 (C3a); 72.4 (2C, C2a, C5a); 67.6 (C4a); 62.0 (C6a).
[0248] Other Signals:
[0249] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 8.08-7.92
(m, 68H, arom. H); 7.61-7.09 (m, 102H, arom. H); 5.45-5.37 (m, 3H,
H2a.beta., H3e); 5.26-5.18 (m, 5H, H2a.alpha., H2e, H4e); 5.11-4.83
(m, 14H, H1b, H2b, H4b, H2e, H4c, H2d, H4d); 4.61-4.35 (m, 10H,
H3a.beta., H5a.alpha., H6a.beta., H6'a.beta., H1c, H1d, H1e);
4.20-3.86 (m, 26H, H6a.alpha., H6'a.alpha., H3b, 116b, H6'b, H3c,
H6c, H6'c, H3d, H6d, H6'd, H5e, H6e, H6'e); 3.80-3.65 (m, 6H, H5b,
H5c, H5d).
[0250] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 166.0,
165.9, 165.8, 165.7, 165.6, 165.4, 164.9, 164.8, 164.7, 164.6,
164.5, 164.3, 164.1, 163.8, 163.6, 163.5 (OCOPh); 133.3, 132.7,
130.0, 129.3, 129.2, 129.1, 129.0, 128.9, 128.7 (quat. arom. C);
133.8, 133.6, 133.4, 133.2, 133.1, 133.0, 132.9, 132.8, 132.6,
129.9, 129.7, 129.6, 129.5, 129.4, 128.6, 128.5, 128.4, 128.3,
128.2, 128.1, 128.0, 127.9 (arom. C); 101.4, 101.3, 101.0, 100.8,
100.7 (8C, C1b, C1c, C1d, C1e); 78.1, 77.5, 77.4, 77.2 (6C, C3b,
C3c, C3d); 73.4, 73.3, 73.2 (4C, 2C2); 72.7, 72.6, 72.5, 72.4 (4C,
C3e, C2); 71.8, 71.7, 71.6, 71.5 (8C, C5b, C5c, C5d, C5e); 71.3
(2C, C2e); 70.3, 70.2 (6C, C4b, C4c, C4d); 69.8 (2C, C4e); 63.5,
63.4, 63.1, 62.7 (9C, C6a.alpha., C6b, C6c).
6.2/ Preparation of
2,3,4,6-tetra-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O--
benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D--
glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D-glucopyranosyl-(1-
.fwdarw.3)-2,4,6-tri-O-benzoyl-.alpha.-D-glucopyranosyl bromide
(Product 3a)
[0251] The perbenzoylated oligosaccharide 2a (444 mg, 0.170 mmol)
is put into solution in dichloromethane (10 mL), then 33% by wt
hydrobromic acid in acetic acid (0.6 mL, 3.417 mmol) was added at
0.degree. C. After stirring for 3.5 hours, the medium is diluted
with dichloromethane then washed with a saturated aqueous solution
of sodium bicarbonate. The organic phase is then dried over
MgSO.sub.4 and concentrated under reduced pressure in order to
produce 435 mg of brominated compound 3a in quantitative
manner.
[0252] Product 3a: colourless oil.
[0253] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm):
Characteristic signals 6.55 (d, 1H, H1a, J.sub.H1a-H2a=4.1 Hz);
5.35 (t, 1H, H4a, J.sub.H3a-H4a=J.sub.H4a-H5a=9.9 Hz); 4.24 (dd,
1H, H6'a, J.sub.H5a-H6'a=4.6 Hz, J.sub.H6a-H6'a=12.4. Hz).
[0254] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm):
Characteristic signals 87.7 (C1a); 76.3 (C3a); 73.4 (C2a); 72.6
(C5a); 67.6 (C4a); 62.0 (C6a).
[0255] Other Signals:
[0256] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 8.92-7.61
(m, 22H, arom. H); 7.48-6.93 (m, 58H, arom. H); 5.27 (t, 1H, H3e,
J.sub.H2e-H3e=J.sub.H3e-H4e=9.7 Hz); 5.09 (dd, 1H, H2e,
J.sub.H1e-H2e=7.9 Hz); 5.08 (t, 1H, H4e, J.sub.H4e-H5e=9.7 Hz);
4.93-4.86 (m, 3H, 2H2, H4); 4.80-4.70 (m, 4H, H2a, H1, 2 H4);
4.50-4.33 (m, 6H, H3a, H5a, H6a, H1e, 2Hp; 4.08-3.94 (m, 3H, H5e,
H3, H6); 3.87-3.70 (m, 9H, 2H3, 7 H6); 3.65-3.53 (m, 3H, H5b, H5c,
H5d).
[0257] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 165.9,
165.8, 165.7, 165.4, 164.9, 164.8, 164.7, 164.6, 164.5, 164.3,
163.8, 163.5 (16C, OCOPh); 133.8, 133.4, 133.2, 133.1, 133.0,
132.9, 132.8, 132.6, 129.9, 129.7, 129.6, 129.5, 129.4, 129.3,
129.2, 129.0, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1,
128.0, 127.9 (arom. C); 101.3, 101.0, 100.8, 100.7 (4C, C1b, C1c,
C1d, C1e); 78.4, 78.1, 77.4 (3C, C3b, C3c, C3d); 73.3, 73.2, 72.7
(3C, C2b, C2c, C2d); 72.5 (C3e); 71.8, 71.7, 71.6, 71.5 (4C, C5b,
C5c, C5d, C5e); 71.3 (C2e); 70.2 (3C, C4b, C4c, C4d); 69.8 (C4e);
63.4, 63.1 (4C, C6b, C6c, C6d, C6e).
6.3/ Preparation of
2,3,4,6-tetra-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O--
benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D--
glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D-glucopyranosyl-(1-
.fwdarw.3)-2,4,6-tri-O-benzoyl-2-D-glu-hex-1-enopyranose (Product
4a)
[0258] The crude product previously obtained (3a: 435 mg, 0.170
mmol) is dissolved in dichloromethane (10 mL) then
1,8-diazabicyclo[5,4,0]undec-7-ene (40 .mu.L, 0.270 mmol) is added.
After stirring for 5.5 hours at ambient temperature, the medium is
diluted with dichloromethane, washed with a 10% aqueous
hydrochloric acid solution, with a saturated aqueous solution of
sodium bicarbonate, then with a saturated aqueous solution of
sodium chloride. The organic phase is dried (MgSO.sub.4),
evaporated and the sought product is obtained after purification on
silica gel [petroleum ether/ethyl acetate (3:2; v/v)]. The target
compound 4a (343 mg) is then isolated with a yield of 81%.
[0259] Product 4a: white solid; Rf (EP/AcOEt, 3:2) 0.4.
[0260] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm):
Characteristic signals 6.63 (s, 1H, H1a); 5.77 (d, 1H, H3a,
J.sub.H3a-H4a=3.3 Hz); 4.73 (dd, 1H, H6a, J.sub.H5a-H6a=9.0 Hz,
J.sub.H6a-H6'a=12.6 Hz); 4.40 (dd, 1H, H6'a, J.sub.H5a-H6'a=3.1
Hz).
[0261] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm):
Characteristic signals 138.2 (C1a); 74.1 (C5a); 72.3 (C4a); 68.7
(C3a); 61.6 (C6a).
[0262] Other Signals:
[0263] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 8.14-7.70
(m, 26H, arom. H); 7.63-7.03 (m, 54H, arom. H); 5.38 (t, 1H, H3e,
J.sub.H2e-H3e=J.sub.H3e-H4e=9.5 Hz); 5.21 (dd, 1H, H2e,
J.sub.H1e-H2e=8.0 Hz); 5.20 (t, 1H, H4e, J.sub.H4e-H5e=9.5 Hz);
5.17 (t, 1H, H2b, J.sub.H1b-H2b=J.sub.H2b-H3b=8.8 Hz); 5.12 (t, 1H,
H4b, J.sub.H3b-H4b=J.sub.H4b-H5b=10.5 Hz); 5.05-4.97 (m, 2H, H4c,
H2d); 4.94 (d, 1H, H1b); 4.94-4.88 (m, 2H, H2c, H4d); 4.71 (d, 1H,
H1e, J.sub.H1c-H2c=6.7 Hz); 4.59 (d, 1H, H1e, J.sub.H1e-H2e=8.0
Hz); 4.58-4.53 (m, 3H, H4a, 145a, H1d); 4.46 (dd, 1H, H6b,
J.sub.H5b-H6b=3.1 Hz, J.sub.H6b-H6'b=12.2 Hz); 4.31 (dd, 1H, H3b);
4.25 (dd, 1H, H6'b, J.sub.H5b-H6'b=6.6 Hz); 4.15-3.87 (m, 9H, H5b,
H3c, H6c, H6'c, H3d, H6d, H6'd, H6e, H6'e); 3.80 (dt, 1H, H5d,
J.sub.H5d-H6d=4.9 Hz, J.sub.H5d-H6'd=9.3 Hz); 3.76-3.68 (m, 21-1,
H5c, H5e).
[0264] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 165.9,
165.8, 165.7, 165.4, 165.3, 165.1, 165.0, 164.8, 164.7, 164.6,
163.9, 163.7 (16C, OCOPh); 133.3, 133.2, 133.0, 132.9, 132.7,
130.1, 130.0, 129.9, 129.7, 129.6, 129.5, 129.3, 129.2, 129.1,
128.9, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0,
127.8 (C2a, arom. C); 101.2 (C1b); 100.9 (C1c); 100.8 (C1e); 100.7
(C1d); 78.2, 78.0, 77.7 (3C, C3b, C3c, C3d); 73.5 (C2c); 73.2
(C2b); 72.8 (C2d); 72.7 (C5b); 72.6 (C3e); 72.1 (C5d); 71.6 (2C,
C5c, C5e); 71.3 (C2e); 70.3 (C4c); 70.0 (C4d); 69.9, 69.8 (2C, C4b,
C4e); 63.5, 63.4, 63.2 (3C, C6c, C6d, C6e); 63.1 (C6b).
6.5/ Preparation of
2,3,4,6-tetra-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O--
benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D--
glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D-glucopyranosyl-(1-
.fwdarw.3)-4,6-di-O-benzoyl-.alpha.-D-glucopyranos-2-ulosyl bromide
(Product 5a)
[0265] Methanol (1.6 .mu.L, 0.040 mmol) is added to the previously
described compound 5a (48 mg, 0.019 mmol) in solution in
dichloromethane (0.5 mL). After stirring for 10 minutes at ambient
temperature, N-bromosuccinimide (10.8 mg, 0.060 mmol) is added at
0.degree. C. Once the reaction has stopped, the medium is diluted
with dichloromethane then washed with an iced saturated aqueous
solution of sodium thiosulphate and with iced water. The organic
phase is then dried over MgSO.sub.4 and concentrated under reduced
pressure in order to produce 5a with a quantitative yield (47 mg,
0.019 mmol).
[0266] Product 5a: colourless oil.
[0267] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm):
Characteristic signals 6.25 (s, 1H, H1a); 5.44 (t, 1H, H4a,
J.sub.H3a-H4a=J.sub.H4a-H5a=10.1 Hz); 5.17 (d, 1H, H3a).
[0268] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm):
Characteristic signals 191.5 (C2a); 83.8 (C1a); 76.3 (C3a); 72.6
(C5a); 69.0 (C4a); 61.7 (C6a).
[0269] Other Signals:
[0270] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 8.06-7.71
(m, 20H, arom. H); 7.61-7.12 (m, 55H, arom. H); 5.39 (t, 1H, H3e,
J.sub.H2e-H3e=J.sub.H3e-H4e=9.5 Hz); 5.22 (dd, 1H, H2e,
J.sub.H1e-H2e=8.0 Hz); 5.21 (t, 1H, H4e, J.sub.H4e-H5e=9.5 Hz);
5.10 (dd, 1H, H2b, J.sub.H1b-H2b=8.2 Hz, J.sub.H2b-H3b=9.3 Hz);
5.06-4.98 (m, 4H, H1b, H4b, H4c, H2d); 4.89 (t, 1H, H4d,
J.sub.H3d-H4d=J.sub.H4d-H5d=9.5 Hz); 4.88 (dd, 1H, H2c,
J.sub.H1c-H2c=7.7 Hz, J.sub.H2c-H3c=8.6 Hz); 4.74 (d, 1H, H1e,
J.sub.H1c-H2c=8.2 Hz); 4.69 (d, 1H, H1e); 4.66-4.55 (m, 3H, H5a,
H6a, H1d); 4.39-4.26 (m, 2H, H3b, H6); 4.24-4.10 (m, 3H, 3H6);
4.05-3.86 (m, 8H, H5b, H3c, H3d, 5H6); 3.82-3.70 (m, 3H, H5c, H5d,
H5e).
[0271] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 167.1,
165.9, 165.8, 165.7, 165.4, 164.9, 164.7, 164.6, 164.3, 164.1,
163.9, 163.6 (15C, OCOPh); 133.6, 133.3, 133.2, 133.1, 133.0,
132.9, 132.8, 132.7, 130.3, 130.0, 129.8, 129.7, 129.6, 129.5,
129.4, 129.3, 129.2, 129.1, 129.0, 128.8, 128.7, 128.6, 128.5,
128.4, 128.3, 128.2, 128.1, 128.0, 127.8, 127.6 (arom. C); 101.0,
100.9, 100.7, 100.6 (4C, C1b, C1c, C1d, C1e); 78.2, 78.0, 77.7 (3C,
C3b, C3c, C3d); 73.6, 73.1, 72.8 (3C, C2b, C2c, C2d); 72.6 (C3e);
71.9, 71.6 (4C, C5b, C5c, C5d, C5e); 71.3 (C2e); 70.1, 69.9, 69.8
(4C, C4b, C4c, C4d, C4e); 63.5, 63.4, 63.1 (4C, C6b, C6c, C6d,
C6e).
6.6/ Preparation of benzyl
2,3,4,6-tetra-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O--
benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D--
glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D-glucopyranosyl-(1-
.fwdarw.3)-6-O-benzoyl-4-deoxy-.beta.-D-gluco-hex-3-enopyranos-2-uloside
(Product 6a)
[0272] Procedure 1:
[0273] Benzyl alcohol (7 .mu.L, 0.068 mmol) and ulosyl bromide 5a
(82 mg, 0.034 mmol) are put into solution in dichloromethane (1.5
mL) in the presence of a molecular sieve. After stirring vigorously
for 5 minutes, triphenylphosphine oxide (19 mg, 0.068 mmol) is
added rapidly to the mixture. After stirring for 4 days at ambient
temperature, the medium is filtered on celite and concentrated
under reduced pressure. The sought product 6a (69 mg; 0.029 mmol)
is obtained after purification on silica gel [petroleum ether/ethyl
acetate (3:2; v/v)] with a yield of 85% in mixture with its
hydrated form.
[0274] Product 6a: colourless oil; Rf (EP/AcOEt, 1:1) 0.5.
[0275] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm):
Characteristic signals 6.26 (d, 1H, H4a, J.sub.H4a-H5a=3.6 Hz);
4.87 (s, 1H, H1a); 4.83 (d, 1H, H7a, J.sub.H7a-H7'a=11.7 Hz);
4.68-4.61 (m, 1H, H5a); 4.60 (d, 1H, H7'a).
[0276] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm):
Characteristic signals 183.5 (C2a); 144.9 (C3a); 126.2 (C4a); 97.7
(C1a); 71.0 (C5a); 70.6 (C7a); 66.1 (C6a).
[0277] Procedure 2:
[0278] Silver triflate (5.2 mg, 0.020 mmol) is added to a solution
of the donor 5a (50 mg, 0.020 mmol) and benzyl alcohol (4.4 .mu.L,
0.040 mmol) in dichloromethane (0.75 mL) at 0.degree. C. After
stirring for 3 hours, the reaction medium is neutralized by the
addition of triethylamine, the silver salts are filtered on celite
and the filtrate is concentrated under vacuum. Purification on
silica gel [petroleum ether/ethyl acetate (3:2; v/v)] makes it
possible to obtain 38 mg (0.016 mmol) of the deoxy compound 6a with
a yield of 75%.
[0279] Other Signals:
[0280] NMR .sup.1H (CDCl.sub.3, 400 MHz) (ppm): 8.10-7.70 (m, 20H,
arom. H); 7.60-7.12 (m, 55H, arom. H); 5.39 (t, 1H, H3e,
J.sub.H2e-H3e=J.sub.H3e-H4e=9.7 Hz); 5.28-5.17 (m, 5H, H1b, H2b,
H4b, H2e, H4e); 5.09-5.02 (m, 4H, H2c, H4c, H2d, H4d); 4.95 (d, 1H,
H1c, J.sub.H1c-H2c=8.0 Hz); 4.63 (d, 1H, H1d, J.sub.H1d-H2d=7.9
Hz); 4.61 (d, 1H, H1e, J.sub.H1e-H2e=8.0 Hz); 4.53-4.40 (m, 3H,
H6a, 2H6); 4.38-4.32 (m, 2H, H3b, 1-16); 4.25-4.18 (m, 2H, H6'a,
H3); 4.15-3.90 (m, 8H, H5b, H3, H5, 5H6); 3.80-3.70 (m, 2H, H5e,
H5).
[0281] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 165.8,
165.7, 165.4, 164.9, 164.6, 164.5, 163.9, 163.8 (14C, OCOPh); 136.1
(quat. arom. COCH.sub.2Ph); 133.3, 133.2, 133.1, 133.0, 132.9,
132.7, 129.9, 129.8, 129.7, 129.6, 129.5, 129.4, 129.3, 129.2,
129.0, 128.8, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0,
127.8 (arom. C); 100.9 (2C, C1d, C1e); 100.1 (C1c); 98.3 (C1b);
78.3, 78.0 (2C, C3c, C3d); 76.8 (C3b); 717 (2C, C2c, C2d); 73.0
(C2b); 72.8 (C5b); 72.6 (C3e); 72.1 (1C, C5); 71.6 (C2e); 71.3 (2C,
C5e, C5); 69.9, 69.8 (3C, C4c, C4d, C4e); 69.3 (C4b); 63.4 (2C,
C6e, C6); 63.1 (2C, C6b, C6).
TABLE-US-00001 HRMS (ESI.sup.+): [M + Na].sup.+
C.sub.135H.sub.11NaO.sub.39 theoretical m/z: 2377.6522 measured
m/z: 2377.6516
6.7/ Preparation of benzyl
2,3,4,6-tetra-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O--
benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D--
glucupyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D-glucopyranosyl-(1-
.fwdarw.3)-6-O-benzoyl-4-deoxy-.beta.-D-manno-hex-3-enopyranosyl
(Product 7a)
[0282] Compound 6a (69 mg, 0.029 mmol) is solubilized in THF (0.7
mL) then L-Selectride (1M solution in THF; 28 .mu.L, 0.028 mmol) is
added at -78.degree. C. After 90 minutes at -78.degree. C., the
reaction is assumed to have stopped and the medium is neutralized
by the addition of a few drops of acetic acid. Once the medium has
returned to ambient temperature, dichloromethane is added and the
organic phase is washed with a 10% aqueous hydrochloric acid
solution, a saturated aqueous solution of sodium bicarbonate and a
saturated aqueous solution of sodium chloride. The desired compound
7a is thus obtained after drying (MgSO.sub.4) and concentration of
the organic phase (68 mg; 0.029 mmol; 100%).
[0283] Product 7a: colourless oil; Rf (EP/AcOEt, 1:1) 0.5.
[0284] NMR .sup.1H (CDCl.sub.3, 500 MHz) .delta. (ppm):
Characteristic signals 4.94 (d, 1H, H4a, J.sub.H4a-H5a=1.1 Hz);
4.86 (d, 1H, H7a, J.sub.H7a-H7'a=12.0 Hz); 4.59 (d, 1H, H7'a); 4.44
(d, 1H, H1a, J.sub.H1a-H2a=2.2 Hz); 4.25-4.20 (m, 2H, H5a,
H6a).
[0285] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm):
Characteristic signals 151.4 (C3a); 104.1 (C4a); 97.1 (C1a); 70.4
(C5a); 70.1 (C7a); 66.2 (C6a); 65.1 (C2a).
[0286] Other Signals:
[0287] NMR .sup.1H (CDCl.sub.3, 500 MHz) .delta. (ppm): 8.03-7.70
(m, 20H, arom. H); 7.61-7.13 (m, 55H, arom. H); 5.39 (t, 1H, H3e,
J.sub.H2e-H3e=J.sub.H3e-H4e=9.6 Hz); 5.30 (t, 1H, H4b,
J.sub.H3b-H4b=J.sub.H4b-H5b=8.3 Hz); 5.22 (dd, 1H, H2e,
J.sub.H1e-H2e=7.9 Hz); 5.20 (t, 1H, H4e, J.sub.H4e-H5e=9.6 Hz);
5.18-5.16 (m, 2H, H1b, H2b); 5.07-4.98 (m, 4H, H2c, H4c, H2d, H4d);
4.85 (d, 1H, Hie, J.sub.H1c-H2c=7.9 Hz); 4.62 (d, 2H, H1d, H1e,
J.sub.H1d-H2d=7.9 Hz); 4.51 (dd, 1H, H6, J.sub.H5-H6=3.8 Hz,
J.sub.H6-H6'=12.3 Hz); 4.36-4.30 (m, 2H, H3b, H6); 4.20-4.14 (m,
2H, H6'a, H3); 4.12-3.90 (m, 10H, H2a, H5b, H3, H5, 6 H6);
3.77-3.70 (m, 2H, H5e, H5).
[0288] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 166.1,
165.9, 165.8, 165.7, 165.4, 164.9, 164.7, 164.3, 163.9, 163.8 (14C,
OCOPh); 136.6 (quat. arom. COCH.sub.2Ph); 133.4, 133.2, 133.1,
133.0, 132.9, 132.7, 129.9, 129.7, 129.6, 129.5, 129.4, 129.3,
129.2, 129.0, 128.8, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1,
128.0, 127.8 (arom. C); 100.9 (2C, C1d, C1e); 100.6 (C1c); 97.3
(C1b); 78.3, 77.9 (2C, C3c, C3d); 77.4 (C3b); 73.5 (1C, C2); 72.9
(2C, C2b, C2); 72.6 (C3e); 72.0 (C5b); 71.6 (3C, C5e, 2C5); 71.3
(C2e); 70.4 (2C, C4c, C4d); 69.8 (C4e); 69.3 (C4b); 63.5, 63.3,
63.1 (4C, C6b, C6c, C6d, C6e).
TABLE-US-00002 HRMS (ESI.sup.+): [M + Na].sup.+
C.sub.135H.sub.112NaO.sub.39 theoretical m/z: 2379.6678 measured
m/z: 2379.6667 [M + K].sup.+ C.sub.135H.sub.112KO.sub.29
theoretical m/z: 2395.6418 measured m/z: 2395.6542
6.8/ Preparation of benzyl
.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(13)-.beta.--
D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-4-deoxy-
-.beta.-D-manno-hex-3-enopyranosyl (Product 8a)
[0289] The compound 7a (65 mg, 0.028 mmol) in a
methanol/dichloromethane mixture (2:1, v/v) then sodium methylate
(1M solution in THF; 0.56 mL, 0.056 mmol) are introduced
successively into a flask. After stirring for 6 hours at ambient
temperature, the medium is neutralized by the addition of Amberlite
IR120-H.sup.+ resin, filtered then concentrated under reduced
pressure. The debenzoylated compound 8a is obtained after
chromatography [dichloromethane/methanol (99:1; v/v)] with a
quantitative yield (25 mg; 0.028 mmol).
[0290] Product 8a: colourless oil.
[0291] NMR .sup.1H (CD.sub.3OD, 400 MHz) .delta. (ppm):
Characteristic signals 5.06 (d, 1H, H4a, J.sub.H4a-H5a=1.5 Hz);
4.82 (d, 1H, H7a, J.sub.H7a-H7'a=12.2 Hz); 4.60 (d, 1H, H7'a); 4.49
(d, 1H, H1a, J.sub.H1a-H2a=1.8 Hz); 4.12-4.08 (m, 1H, H5a); 3.87
(d, 1H, H2a).
[0292] NMR .sup.13C (CD.sub.3OD, 100 MHz) .delta. (ppm):
Characteristic signals 153.5 (C3a); 103.8 (C4a); 99.8 (C1a); 71.2
(C7a); 67.2 (C2a); 65.6 (C6a).
[0293] Other Signals:
[0294] NMR .sup.1H (CD.sub.3OD, 400 MHz) .delta. (ppm): 7.35-7.16
(m, 5H, arom. H); 4.71 (d, 1H, H1b, J.sub.H1b-H2b=7.5 Hz); 4.54 (d
1H, H1, J.sub.H1-H2=8.0 Hz); 4.53 (d, 1H, H1, J.sub.H1-H2=7.8 Hz);
4.46 (d, 1H, H1e, J.sub.H1e-H2e=7.8 Hz); 3.80-3.73 (m, 4H, 4H6);
3.60-3.43 (m, 10H, H6a, H6'a, H2b, 3H3, 4 H6); 3.40-3.32 (m, 2H,
H2c, H2d); 3.30-3.17 (m, 8H, H3e, 3H4, 4 H5); 3.16 (dd, 1H, H2e,
J.sub.H2e-H3e=9.3 Hz); 3.15 (t, 1H, H4e,
J.sub.H3e-H4e=J.sub.H4e-H5e=9.8 Hz).
[0295] NMR .sup.13C (CD.sub.3OD, 100 MHz) .delta. (ppm): 138.9
(quat. arom. COCH.sub.2Ph); 129.6, 129.4, 128.9 (arom. C); 105.1,
104.6 (3C, C1c, C1d, C1e); 100.5 (C1b); 87.4, 87.2, 87.1 (3C, C3b,
C3c, C3d); 78.1, 77.8, 77.7 (5C, C3e, C5b, C5c, C5d, C5e); 75.5
(C2e); 75.0, 74.9 (3C, C5a, C2c, C2d); 74.0 (C2b); 71.5 (C4e);
70.0, 69.9 (3C, C4b, C4c, C4d); 62.6, 62.5 (4C, C6b, C6c, C6d,
C6e).
6.9/ Preparation of
.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-4-deoxy-(4)-D-mannopyranose (Product Ca)
[0296] After dissolution of compound 8a (24 mg, 0.027 mmol) in
methanol, palladium acetate (25 mg, 0.111 mmol) is added to the
medium. After stirring for 7 days under a hydrogen atmosphere at
ambient temperature, the medium is filtered then concentrated under
reduced pressure. Compound Ca is obtained after purification by
Sephadex G-10 steric exclusion (eluent: water) and freeze-drying of
the collected fractions (21 mg; 0.027 mmol; 100%).
[0297] Ca: white foam; Rf (AcOEt/iPrOH/H.sub.2O, 3:2:2) 0.2.
[0298] NMR .sup.1H (D.sub.2O, 400 MHz) .delta. (ppm):
Characteristic signals 5.12 (m, 1H, H1a.alpha.); 1.72-1.60 (m, 2H,
H4a, H4'a).
[0299] NMR .sup.13C (D.sub.2O, 100 MHz) .delta. (ppm):
Characteristic signals 96.0 (C1a); 67.2 (C2a); 61.0 (C6a); 28.8
(C4a).
[0300] Other Signals:
[0301] NMR .sup.1H (D.sub.2O, 400 MHz) .delta. (ppm): 4.68 (d, 2H,
2H1, J.sub.H1-H2=8.2 Hz); 4.64 (d, 2H, 2H1, J.sub.H1-H2=8.0 Hz);
3.86-3.73 (m, 5H, H6a, H6b, H6c, H6d, H6e); 3.71-3.56 (m, 10H, H2a,
H5a, H6'a, H3b, H6'b, H3c, H6'c, H3d, H6'd, H6'e); 3.55-3.33 (m,
13H, H3a, H5a, H2b, H4b, H5b, H2c, H4c, H5c, H2d, H4d, H5d, H3e,
H5e); 3.31 (t, 1H, H4e, J.sub.H3e-H4e=J.sub.H4e-H5e=9.3 Hz); 3.24
(dd, 1H, H2e, J.sub.H1e-H2e=8.0 Hz, J.sub.H2e-H3e=9.3 Hz).
[0302] NMR .sup.13C (D.sub.2O, 100 MHz) .delta. (ppm): 103.1, 102.8
(4C, C1b, C1c, C1d, C1e); 84.5, 84.4 (4C, C3a, C3b, C3c, C3d);
76.3, 75.9, 75.8 (6C, C5a, C5b, C5c, C5d, C3e, C5e); 73.8, 73.6
(5C, C2a, C2b, C2c, C2d, C2e); 69.9 (C4e); 64.4 (3C, C4b, C4c,
C4d); 61.0 (4C, C6b, C6c, C6d, C6e).
TABLE-US-00003 HRMS (ESI.sup.+): [M + Na].sup.+
C.sub.30H.sub.52NaO.sub.25 theoretical m/z: 835.2695 measured m/z:
835.2694
Example 7
[0303] The product 1b is prepared according to the procedure
described in the Patent Application FR2804684, according to the
procedure of Example 6.
7.1/ Preparation of
2,3,4,6-tetra-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O--
benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D--
glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D-glucopyranosyl-(1-
.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-1,2,4,-
6-tetra-O-benzoyl-(.alpha.,.beta.)-D-glucopyranose (Product 2b)
[0304] The oligosaccharide 2a (200 mg, 0.202 mmol) is dissolved in
pyridine (20 mL). The reaction medium is then cooled down to
0.degree. C. then benzoyl chloride (5.2 mL, 44.4 mmol) is added.
After stirring for 2 days at ambient temperature, the medium is
evaporated, dissolved in dichloromethane then washed with a 10%
aqueous hydrochloric acid solution, with a saturated solution of
sodium bicarbonate and a saturated solution of sodium chloride
until neutrality is reached. The organic phase is then dried over
MgSO.sub.4 and evaporated. The residue is purified by silica gel
chromatography [petroleum ether/ethyl acetate (1:1; v/v)] in order
to produce the perbenzoylated derivative 2a in the form of a
mixture of anomers .alpha./.beta.:1/1 with a yield of 85% (545 mg,
0.178 mmol).
[0305] Product 2b: colourless oil; Rf (EP/AcOEt, 1:1) 0.4.
[0306] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm):
Characteristic signals for 2b.alpha. 6.60 (d, 1H, H1a,
J.sub.H1a-H2a=3.8 Hz); 5.42 (t, 1H, H4a,
J.sub.H3a-H4a=J.sub.H4a-H5a=9.8 Hz); Characteristic signals for
2b.beta. 6.02 (d, 1H, H1a, J.sub.H1a-H2a=7.1 Hz); 5.52 (t, 1H, H4a,
J.sub.H3a-H4a=J.sub.H4a-H5a=9.0 Hz); 4.23-4.18 (m, 1H, H5a).
[0307] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm):
Characteristic signals for 2b.alpha. 89.7 (C1a); 76.3 (C3a); 72.7
(C2a); 70.2 (C5a); 68.8 (C4a); Characteristic signals for 2b.beta.
92.0 (C1a); 78.3 (C3a); 72.9 (C5a); 72.5 (C2a); 68.8 (C4a); 62.7
(C6a).
[0308] Other Signals:
[0309] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 8.01-7.65
(m, 60H, arom. H); 7.55-7.01 (m, 140H, arom. H); 5.36-5.30 (m, 3H,
H2a.beta., H3f); 5.20-5.12 (m, 5H, H2a.alpha., H2f, H4f); 5.07-4.89
(m, 8H, 2H2, 2 H4); 4.88-4.72 (m, 10H, H1b, 2H2, 2 H4); 4.61-4.30
(m, 13H, H3a.alpha., H3a.beta., H5a.alpha., H6a.beta., H6'a.beta.,
H1c, H1d, H1e, H1f); 4.12-3.78 (m, 32H, H6a.alpha., H6'a.alpha.,
H3b, H6b, H6'b, H3c, H6c, H3d, H6d, H6'd, H3e, H6e, H6'e, H5f, H6f,
H6'f); 3.73-3.56 (m, 8H, H5b, H5c, H5d, H5e).
[0310] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 166.0,
165.9, 165.8, 165.7, 165.4, 164.9, 164.7, 164.6, 164.4, 164.3,
164.1, 163.8, 163.6, 163.5 (OCOPh); 133.5, 133.2, 133.1, 133.0,
132.8, 132.6, 130.0, 129.9, 129.7, 129.6, 129.5, 129.4, 129.3,
129.2, 129.1, 129.0, 128.9, 128.7; 128.6, 128.5, 128.4, 128.2,
128.0, 127.9 (arom. C); 101.4, 101.0, 100.9, 100.8, 100.7 (10C,
C1b, C1c, C1d, C1e, C1f); 78.3, 78.2, 78.1, 77.9 (8C, C3b, C3c,
C3d, C3e); 73.3, 73.2, 73.1 (6C, 3C2); 72.9 (2C, C2); 72.5 (C3f);
72.1 (C5f); 71.7, 71.6 (8C, C5b, C5c, C5d, C5e); 71.3 (2C, C2f);
70.2 (4C, 2C4); 70.0 (4C, 2C4); 69.8 (2C, C4f); 63.7, 63.4, 63.1
(11C, C6a.alpha., C6b, C6c, C6d, C6e, C6f).
TABLE-US-00004 HRMS (ESI.sup.+): [M + Na].sup.+
C.sub.176H.sub.142NaO.sub.51 theoretical m/z: 3093.8416 measured
m/z: 3093.8418 [M + K].sup.+ C.sub.176H.sub.142KO.sub.51
theoretical m/z: 3109.8155 measured m/z: 3109.8119
7.2/ Preparation of
2,3,4,6-tetra-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O--
benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D--
glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D-glucopyranosyl-(1-
.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6--
tri-O-benzoyl-.alpha.-D-glucopyranosyl bromide (Product 3b)
[0311] The bromination of compound 2b (643 mg, 0.209 mmol) is
carried out as described for 3a in dichloromethane (15 mL) in the
presence of hydrobromic acid (1.1 mL, 6.364 mmol) in order to
produce 633 mg of brominated compound 3b in a quantitative
manner.
[0312] Product 3b: Colourless Oil.
[0313] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm):
Characteristic signals 6.68 (d, 1H, H1a, J.sub.H1a-H2a=3.8 Hz);
5.48 (t, 1H, H4a, J.sub.H3a-H4a=J.sub.H4a-H5a=9.7 Hz); 4.82 (dd,
1H, H2a, J.sub.H2a-H3a=9.7 Hz); 4.61 (t, 1H, H3a); 4.60 (dd, 1H,
H6a, J.sub.H5a-H6a=3.8 Hz, J.sub.H6a-H6'a=12.2 Hz); 4.55-4.51 (m,
1H, H5a); 4.37 (dd, 1H, H6'a, J.sub.H5a-H6'a=4.2 Hz).
[0314] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm):
Characteristic signals 87.7 (C1a); 76.3 (C3a); 73.4 (C2a); 72.7
(C5a); 67.6 (C4a); 62.0 (C6a).
[0315] Other Signals:
[0316] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 8.00-7.71
(m, 26H, arom. H); 7.61-7.03 (m, 69H, arom. H); 5.39 (t, 1H, H3f,
J.sub.H2f-H3f=J.sub.H3f-H4f=9.7 Hz); 5.22 (dd, 1H, H2f,
J.sub.H1f-H2f=7.8 Hz); 5.21 (t, 1H, H4f, J.sub.H4f-H5f=9.7 Hz);
5.05-4.97 (m, 4H, 3H2, H4); 4.94 (t, 1H, H4, J.sub.H4-H5=9.3 Hz);
4.88 (d, 1H, H1, J.sub.H1-H2=8.2 Hz); 4.87-4.77 (m, 3H, H2, 2 H4);
4.55 (d, 1H, H1f); 4.49 (d, 1H, H1, J.sub.H1-H2=7.7 Hz); 4.42 (d,
1H, H1, J.sub.H1-H2=7.5 Hz); 4.40 (d, 1H, H1, J.sub.H1-H2=7.5 Hz);
4.19-4.12 (m, 2H, H3, H6); 4.08-3.81 (m, 12H, 3H3, 9 H6); 3.77-3.71
(m, 1H, H5f); 3.71-3.60 (m, 4H, H5b, H5c, H5d, H5e).
[0317] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 165.9,
165.8, 165.7, 165.4, 164.9, 164.8, 164.7, 164.6, 164.5, 164.3,
163.8, 163.5 (19C, OCOPh); 133.8, 133.4, 133.2, 133.1, 133.0,
132.8, 132.6, 129.9, 129.7, 129.6, 129.5, 129.4, 129.3, 129.2,
129.1, 129.0, 128.9, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2,
128.1, 128.0, 127.9 (arom. C); 101.3, 101.0, 100.8, 100.7 (5C, C1b,
C1c, C1d, C1e, C1f); 78.4, 78.1, 77.8, 77.4 (4C, C3b, C3c, C3d,
C3e); 73.3, 73.2, 72.9 (4C, C2b, C2c, C2d, C2e); 72.5 (C3f); 71.8,
71.6 (5C, C5b, C5c, C5d, C5e, C5f); 71.3 (C2f); 70.2 (4C, C4b, C4c,
C4d, C4e); 69.8 (C4f); 63.5, 63.4, 63.1 (5C, C6b, C6c, C6d, C6e,
C6f).
7.3/ Preparation of
2,3,4,6-tetra-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O--
benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D--
glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D-glucopyranosyl-(1-
.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6--
tri-O-benzoyl-2-D-glu-hex-1-enopyranose (Product 4b)
[0318] Compound 3b (550 mg, 0.181 mmol) is involved in an
elimination reaction following the same procedure as that described
for 4a in dichloromethane (10 mL) in the presence of DBU (35 .mu.L,
0.235 mmol). Purification on a silica gel column [petroleum
ether/ethyl acetate (3:2; v/v)] makes it possible to obtain 458 mg
of the desired product 4b with a yield of 85%.
[0319] 4b: white solid; Rf (EP/AcOEt, 1:1) 0.5.
[0320] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm):
Characteristic signals 6.54 (s, 1H, H1a); 5.69 (d, 1H, H3a,
J.sub.H3a-H4a=1.6 Hz); 4.64 (dd, 1H, H6a, J.sub.H5a-H6a=8.8 Hz,
J.sub.H6a-H6'a=12.4 Hz); 4.32 (dd, 1H, H6'a, J.sub.H5a-H6'a=2.4
Hz).
[0321] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm):
Characteristic signals 138.2 (C1a); 74.1 (C5a); 72.3 (C4a); 68.6
(C3a); 61.6 (C6a).
[0322] Other Signals:
[0323] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 7.95-7.62
(m, 25H, arom. H); 7.51-6.93 (m, 70H, arom. H); 5.29 (t, 1H, H3f,
J.sub.H2f-H3f=J.sub.H3f-H4f=9.7 Hz); 5.12 (dd, 1H, H2f,
J.sub.H1f-H2f=8.0 Hz); 5.11 (t, 1H, H4f, J.sub.H4f-H5f=9.7 Hz);
5.10-5.12 (m, 1H, H2b, H4b); 4.96-4.80 (m, 4H, H1, 2 H2, H4);
4.78-4.71 (m, 3H, H2, 2 H4); 4.60 (d, 1H, H1, J.sub.H1-H2=8.0 Hz);
4.52-4.44 (m, 3H, H4a, H5a, H1); 4.42-4.35 (m, 3H, 2H1, H6,
J.sub.H1-H2=8.0 Hz); 4.07-4.01 (m, 1H, H5b); 3.98 (dd, 1H, H6,
J.sub.H5-H6=3.5 Hz, J.sub.H6-H6'=11.9 Hz); 3.93-3.75 (m, 1H, 3H3c,
8H6); 3.74-3.55 (m, 4H, H5c, H5d, H5e, H5f).
[0324] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 165.8,
165.7, 165.4, 165.2, 165.1, 164.9, 164.7, 164.6, 163.8, 163.7,
163.6 (19C, OCOPh); 133.3, 133.2, 133.1, 133.0, 132.9, 132.8,
132.7, 132.6, 129.9, 129.8, 129.7, 129.6, 129.5, 129.4, 129.3,
129.2, 129.1, 129.0, 128.8, 128.6, 128.5, 128.4, 128.3, 128.2,
128.1, 128.0, 127.9, 127.7 (C2a, arom. C); 101.2 (C1b); 100.9,
100.8, 100.7 (4C, C1c, C1d, C1e, C1f); 78.2, 78.0 (4C, C3b, C3c,
C3d, C3e); 73.3 (1C, C2); 73.1 (C2b); 73.0 (C2); 72.7 (C5b); 72.6
(2C, C3f, C2); 72.0, 71.8, 71.6 (4C, C5c, C5d, C5e, C5f); 71.3
(C2f); 70.2, 70.1, 69.8, 69.7 (5C, C4b, C4c, C4d, C4e, C4f); 63.5,
63.3, 63.1 (5C, C6b, C6c, C6d, C6e, C6f).
TABLE-US-00005 HRMS (ESI.sup.+): [M + Na].sup.+
C.sub.169H.sub.136NaO.sub.49 theoretical m/z: 2971.8048 measured
m/z: 2971.8034 [M + K].sup.+ C.sub.169H.sub.136KO.sub.49
theoretical m/z: 2987.7787 measured m/z: 2987.7811
7.4/ Preparation of
2,3,4,6-tetra-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O--
benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D--
glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D-glucopyranosyl-(1-
.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-4,6-di-
-O-benzoyl-.alpha.-D-glucopyranos-2-ulosyl bromide (Product 5b)
[0325] Compound 5b was obtained following the same procedure as
that described for the preparation of 5a from 4b (124 mg, 0.042
mmol) in 1.5 mL of dichloromethane, NBS (22.4 mg, 0.126 mmol) and
methanol (3.4 .mu.L, 0.084 mmol) with a quantitative yield (123 mg,
0.042 mmol).
[0326] Product 5b: colourless oil.
[0327] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm):
Characteristic signals 6.25 (s, 1H, H1a); 5.44 (t, 1H, H4a,
J.sub.H3a-H4a=J.sub.H4a-H5a=10.2 Hz); 5.16 (d, 1H, H3a).
[0328] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 1
Characteristic signals 91.5 (C2a); 83.9 (C1a); 76.3 (C3a); 72.6
(C5a); 69.0 (C4a); 61.7 (C6a).
[0329] Other Signals:
[0330] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 8.09-7.70
(m, 25H, arom. H); 7.60-6.84 (m, 65H, arom. H); 5.37 (t, 1H, H3f,
J.sub.H2f-H3f=J.sub.H3f-H4f=9.7 Hz); 5.20 (dd, 1H, H2f,
J.sub.H1f-H2f=8.0 Hz); 5.19 (t, 1H, H4f, J.sub.H4f-H5f=9.7 Hz);
5.10 (dd, 1H, H2b, J.sub.H1b-H2b=8.1 Hz, J.sub.H2b-H3b=9.1 Hz);
5.07-4.96 (m, 4H, H 1b, H2, 2 H4); 4.92-4.78 (m, 4H, 2H2, 2 H4);
4.73 (d, 1H, H1, J.sub.H1-H2=8.0 Hz); 4.66 (d, 1H, H1,
J.sub.H1-H2=8.0 Hz); 4.65-4.57 (m, 2H, H5a, H6); 4.54 (d, 1H, H1,
J.sub.H1-H2=8.0 Hz); 4.49 (d, 1H, H1, J.sub.H1-H2=7.7 Hz);
4.47-4.41 (m, 1H, H6); 4.35 (dd, 1H, H6, J.sub.H5-H6=4.9 Hz,
J.sub.H6-H6'=12.4 Hz); 4.28 (t, 1H, H3b, J.sub.H3b-H4b=9.1 Hz);
4.19 (dd, 1H, H6, J.sub.H5-H6=3.5 Hz, J.sub.H6-H6'=12.2 Hz); 4.12
(dd, 1H, H6, J.sub.H5-H6=6.2 Hz, J.sub.H6-H6'=12.2 Hz); 4.05-3.81
(m, 11H, H5b, H3c, H3d, H3e, 7H6); 3.80-3.62 (m, 4H, H5c, H5d, H5e,
H5f).
[0331] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 165.9,
165.8, 165.7, 165.4, 164.9, 164.7, 164.6, 164.3, 163.8, 163.6 (18C,
OCOPh); 133.6, 133.3, 133.2, 133.1, 133.0, 132.9, 132.6, 130.0,
129.8, 129.7, 129.6, 129.5, 129.4, 129.3, 129.2, 129.1, 129.0,
128.8, 128.6, 128.4, 128.3, 128.2, 128.1, 127.9 (arom. C); 101.0,
100.9, 100.7, 100.6 (5C, C1b, C1c, C1d, C1e, C1f); 78.3, 78.2,
78.1, 78.0 (4C, C3b, C3c, C3d, C3e); 73.5, 73.1, 73.0, 72.8 (4C,
C2b, C2c, C2d, C2e); 72.6 (C3f); 71.9, 71.8, 71.6 (5C, C5b, C5c,
C5d, C5e, C5f); 71.3 (C2f); 70.2, 70.1, 70.0, 69.8 (5C, C4b, C4c,
C4d, C4e, C4f); 63.6, 63.5, 63.4, 63.3, 63.2 (5C, C6b, C6c, C6d,
C6e, C6f).
7.5/ Preparation of benzyl
2,3,4,6-tetra-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O--
benzoyl-D-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D-gluco-
pyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwda-
rw.3)-2,4,6-tri-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-6-O-benzoyl-
-4-deoxy-.beta.-D-gluco-hex-3-enopyranos-2-uloside (Product 6b)
[0332] Benzyl alcohol (7 .mu.L, 0.068 mmol) and ulosyl bromide 5b
(183 mg, 0.063 mmol) are put into solution in dichloromethane (1.5
mL) in the presence of a molecular sieve. After stirring vigorously
for 5 minutes, triphenylphosphine oxide (19 mg, 0.068 mmol) is
rapidly added to the mixture. After stirring for 4 days at ambient
temperature, the medium is filtered on celite, concentrated under
reduced pressure and purified by chromatography on silica
[petroleum ether/ethyl acetate (3:2; v/v)]. After evaporation of
the solvents, the crude product thus obtained is dissolved in
benzene (8 mL), then water (200 .mu.L, 11.1 mol) and sodium
bicarbonate (200 mg, 2.381 mmol) are added. The medium is then
taken to 80.degree. C. for 1.5 hours. After returning to ambient
temperature, the suspension is dried over MgSO4, filtered and
concentrated under vacuum in order to produce the product 6b (120
mg; 0.029 mmol) with an overall yield of 67%.
[0333] Product 6b: colourless oil; Rf (EP/AcOEt, 1:1) 0.5.
[0334] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm):
Characteristic signals 6.26 (d, 1H, H4a, J.sub.H4a-H5a=3.5 Hz);
4.87 (s, 1H, H1a); 4.83 (d, 1H, H7a, J.sub.H7a-H7'a=11.7 Hz);
4.71-4.65 (m, 1H, H5a); 4.60 (d, 1H, H7'a).
[0335] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm):
Characteristic signals 183.6 (C2a); 144.9 (C3a); 126.3 (C4a); 97.7
(C1a); 71.0 (C5a); 70.6 (C7a); 66.1 (C6a).
[0336] Other Signals:
[0337] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 8.06-7.71
(m, 25H, arom. H); 7.60-7.12 (m, 65H, arom. H); 5.37 (t, 1H, H3f,
J.sub.H2f-H3f=J.sub.H3f-H4f=9.5 Hz); 5.30-5.16 (m, 5H, H1b, H2b,
H2f, H4b, H4f); 5.07-4.94 (m, 6H, H2c, H2d, H2e, H4c, H4d, H4e);
4.90 (d, 1H, H1c, J.sub.H1c-H2c=8.0 Hz); 4.57 (d, 1H, H1,
J.sub.H1-H2=8.2 Hz); 4.55 (d, 2H, H1, J.sub.H1-H2=8.0 Hz);
4.54-4.43 (m, 3H, H6a, 2H6); 4.38-4.32 (m, 2H, H3b, H6); 4.24-4.20
(m, 2H, H3, H6'a); 4.16-4.04 (m, 4H, H6'a, H3, H5, H6); 4.02-3.82
(m, 8H, H3, 2 H5, 5 H6); 3.76-3.64 (m, 3H, H5f, 2H5).
[0338] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 165.9,
165.8, 165.7, 165.4, 164.9, 164.7, 164.6, 164.4, 163.8, 163.6 (15C,
OCOPh); 136.1 (quat. arom. COCH.sub.2Ph); 133.3, 133.1, 133.0,
132.9, 132.6, 130.0, 129.8, 129.7, 129.6, 129.5, 129.4, 129.3,
129.2, 129.1, 129.0, 128.8, 128.7, 128.6, 128.5, 128.4, 128.2,
128.1, 128.0 (arom. C); 101.0, 100.9, 100.8 (3C, C1d, C1e, C1f);
100.2 (C1c); 98.4 (C1b); 78.3, 78.2, 78.1 (3C, C3c, C3d, C3e); 77.6
(C3b); 73.3, 73.0, 72.9 (5C, C2b, C5b, C2c, C2d, C2e); 72.6 (C3f);
72.1, 72.0, 71.8 (3C, C5); 71.6 (C2f); 71.3 (2C, C5f, C5); 70.1,
70.0 (3C, C4c, C4d, C4e); 69.8 (C4f); 69.3 (C4b); 63.5, 63.4 (3C,
C6f, 2C6); 63.2, 63.1 (2C, C6b, C6).
TABLE-US-00006 HRMS (ESI.sup.+): [M + Na].sup.+
C.sub.162H.sub.132NaO.sub.47 theoretical m/z: 2851.7837 measured
m/z: 2852.7830 [M + K].sup.+ C.sub.162H.sub.132KO.sub.47
theoretical m/z: 2867.7576 measured m/z: 2867.7508
7.6/ Preparation of benzyl
2,3,4,6-tetra-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O--
benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D--
glucopyranosyl-(1.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D-glucopyranosyl-(1-
.fwdarw.3)-2,4,6-tri-O-benzoyl-.beta.-D-glucopyranosyl-(1.fwdarw.3)-6-O-be-
nzoyl-4-deoxy-.beta.-D-manno-hex-3-enopyranosyl (Product 7b)
[0339] The reduction of compound 6b (104 mg, 0.036 mmol) is carried
out following the same procedure as that described for 7a in the
presence of L-Selectride (1M solution in THF; 50 .mu.L, 0.050 mmol)
in THF (1 mL). The compound 7b (105 mg; 0.036 mmol) is thus
obtained in a quantitative manner after treatments.
[0340] Product 7b: colourless oil; Rf (EP/AcOEt, 1:1) 0.5.
[0341] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm):
Characteristic signals 4.96 (d, 1H, H4a, J.sub.H4a-H5a<1.0 Hz);
4.60 (d, 1H, H7'a, J.sub.H7a-H7'a=11.7 Hz); 4.45 (d, 1H, H1a,
J.sub.H1a-H2a=2.2 Hz); 4.28-4.22 (m, 2H, H5a, H6a).
[0342] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm):
Characteristic signals 151.4 (C3a); 104.1 (C4a); 97.0 (C1a); 70.3
(C5a); 70.0 (C7a); 66.2 (C6a); 65.1 (C2a).
[0343] Other Signals:
[0344] NMR .sup.1H (CDCl.sub.3, 400 MHz) .delta. (ppm): 8.04-7.73
(m, 25H, arom. H); 7.60-7.10 (m, 65H, arom. H); 5.39 (t, 1H, H3f,
J.sub.H2f-H3f=J.sub.H3f-H4f=9.7 Hz); 5.34 (t, 1H, H4b,
J.sub.H3b-H4b=J.sub.H4b-H5b=8.6 Hz); 5.22 (dd, 1H, H2f,
J.sub.H1f-H2f=7.7 Hz); 5.21 (t, 1H, H4f, J.sub.H4f-H5f=9.7 Hz);
5.19 (dd, 1H, H2b, J.sub.H1b-H2b=7.1 Hz, J.sub.H2b-H3b=9.5 Hz);
5.18 (d, 1H, H1b); 5.06-4.97 (m, 3H, H2, 2 H4); 4.90-4.81 (m, 4H,
H7a, 2H2, H4); 4.58 (d, 3H, 3H1, J.sub.H1-H2=7.7 Hz); 4.53 (dd, 1H,
H6, J.sub.H5-H6=3.3 Hz, J.sub.H6-H6'=12.2 Hz); 4.49 (d, 1H, H1,
J.sub.H1-H2=8.0 Hz); 4.39-4.32 (m, 2H, H3b, H6); 4.20-4.06 (m, 4H,
H6'a, H3, H5, H6); 4.00-3.83 (m, 12H, H2a, H5f, 3H3, 7 H6);
3.76-3.71 (m, 4H, 4H5).
[0345] NMR .sup.13C (CDCl.sub.3, 100 MHz) .delta. (ppm): 166.1,
165.9, 165.8, 165.7, 165.4, 164.9, 164.7, 164.6, 164.5, 164.4,
164.2, 163.8, 163.6 (17C, OCOPh); 136.5 (quat. arom. COCH.sub.2Ph);
133.4, 133.2, 133.1, 133.0, 132.9, 132.6, 129.9, 129.8, 129.7,
129.6, 129.5, 129.4, 129.3, 129.2, 129.1, 129.0, 128.9, 128.7,
128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9 (arom. C);
101.0, 100.9, 100.7, 100.6 (4C, C1c, C1d, C1e, C1f); 97.3 (C1b);
78.2 (3C, C3c, C3d, C3e); 77.5 (C3b); 73.4, 73.1 (3C, C2c, C2d,
C2e); 72.9 (C2b); 72.5 (C30; 71.9 (C5b); 71.7 (1C, C5); 71.6 (3C,
C5f, 2C5); 71.2 (C2f); 70.1 (2C, C4); 69.7 (C4f); 69.3, 69.2 (2C,
C4b, C2); 63.4, 63.3, 63.1 (5C, C6b, C6c, C6d, C6e, C6f).
TABLE-US-00007 HRMS (ESI.sup.+): [M + Na].sup.+
C.sub.162H.sub.134NaO.sub.47 theoretical m/z: 2853.7993 measured
m/z: 2853.7994 [M + K].sup.+ C.sub.162H.sub.134KO.sub.47
theoretical m/z: 2869.7732 measured m/z: 2869.7650
7.7/ Preparation of benzyl
.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-.beta.-D-glucopyranosyl-(1.fwdarw.3)-4-deoxy-.beta.-D-manno-hex-3-enopyra-
nosyl (Product 8b)
[0346] Compound 7b (105 mg, 0.036 mmol) is involved in a
debenzoylation reaction following the same procedure as that
described for 8a in a dichloromethane/methanol mixture (3:2; v/v)
(2.5 mL) in the presence of MeONa (0.1 M solution in MeOH; 0.71 mL,
0.71 mmol). Purification on a silica gel column [ethyl
acetate/methanol (3:2; v/v)] makes it possible to obtain 8b (38 mg)
with a quantitative yield.
[0347] Product 8b: colourless oil.
[0348] NMR .sup.1H (CD.sub.3OD, 400 MHz) .delta. (ppm):
Characteristic signals 5.06 (d, 1H, H4a, J.sub.H4a-H5a=1.6 Hz);
4.82 (d, 1H, H7a, J.sub.H7a-H7'a=11.9 Hz); 4.60 (d, 1H, H7'a); 4.49
(d, 1H, H1a, J.sub.H1a-H2a=1.8 Hz); 4.12-4.08 (m, 1H, H5a); 3.88
(d, 1H, H2a).
[0349] NMR .sup.13C (CD.sub.3OD, 100 MHz) .delta. (ppm):
Characteristic signals 153.5 (C3a); 103.9 (C4a); 99.8 (C1a); 71.2
(C7a); 67.2 (C2a); 65.6 (C6a).
[0350] Other Signals:
[0351] NMR .sup.1H (CD.sub.3OD, 400 MHz) .delta. (ppm): 7.30-7.17
(m, 5H, arom. H); 4.71 (d, 1H, H1b, J.sub.H1b-H2b=7.7 Hz); 4.55 (d,
3H, H1c, H1d, H1e, J.sub.H1-H2=7.8 Hz); 4.47 (d, 1H, H1f,
J.sub.H1f-H2f=8.0 Hz); 3.81-3.72 (m, 5H, 5H6); 3.62-3.44 (m, 12H,
116a, H6'a, H2b, 3H3, 5 H6); 3.40-3.34 (m, 3H, H2c, H2d, H2e);
3.32-3.14 (m, 12H, H2f, H3f, H4f, 4H4, 5 H5).
[0352] NMR .sup.13C (CD.sub.3OD, 100 MHz) .delta. (ppm): 138.9
(quat. arom. COCH.sub.2Ph); 129.4, 129.3, 128.9, 128.7 (arom. C);
105.2, 104.7 (4C, C1c, C1d, C1e, C1f); 100.5 (C1b); 87.4, 87.3,
87.1 (4C, C3b, C3c, C3d, C3e); 78.1, 77.8 (6C, C3f, C5b, C5c, C5d,
C5e, C5f); 75.5 (C2f); 75.0, 74.9 (4C, C2c, C2d, C2e, C5a); 74.0
(C2b); 71.5 (C4f); 70.0, 69.9 (4C, C4b, C4c, C4d, C4e); 62.6, 62.5
(5C, C6b, C6c, C6d, C6e, C6f).
7.8/ Preparation of
.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-.beta.-D-glucopyranosyl-(1.fwdarw.3)-4-deoxy-(.alpha.,.beta.)-D-mannopyra-
nose (Product Cb)
[0353] Compound 8b (60 mg, 0.056 mmol) is reduced and debenzylated
following the same procedure as that described for Ca, namely by
hydrogenolysis in the presence of palladium acetate (60 mg, 0.267
mmol). Purification on a Sephadex G-10 column leads in a
quantitative manner to the desired product Cb in .alpha./.beta.
mixture where compound .alpha. is by far the majority
component.
[0354] Product Cb: White Foam.
[0355] NMR .sup.1H (D.sub.2O, 400 MHz) .delta. (ppm):
Characteristic signals 5.14 (d, 1H, H1a.alpha., J.sub.H1a-H2a=3.3
Hz); 1.75-1.60 (m, 2H, H4a).
[0356] NMR .sup.13C (D.sub.2O, 100 MHz) .delta. (ppm):
Characteristic signals 96.0 (C1a.alpha.); 61.0 (C6a); 26.0
(C4a).
[0357] Other Signals:
[0358] NMR .sup.1H (D.sub.2O, 400 MHz) .delta. (ppm): 4.70 (d, 3H,
H1, J.sub.H1-H2=8.0 Hz); 4.66 (d, 2H, H1, J.sub.H1-H2=8.2 Hz);
3.86-3.78 (m, 6H, H6a, H6b, H6c, H6d, H6e, H6f); 3.73-3.60 (m, 11H,
H3a, H3b, H3c, H3d, H3e, H6'a, H6'b, H6'c, H6'd, H6'e, H6'f);
3.50-3.36 (m, 16H, H2a, H2b, H2c, H2d, H2e, H3f, H4b, H4c, H4d,
H4e, H5a, H5b, H5e, H5d, H5e, H5f); 3.31 (t, 1H, H4f,
J.sub.H3f-H4f=J.sub.H4f-H5f=9.3 Hz); 3.26 (dd, 1H, H2f,
J.sub.H1f-H2f=8.0 Hz, J.sub.H2f-H3f=9.0 Hz).
[0359] NMR .sup.13C (D.sub.2O, 100 MHz) .delta. (ppm): 103.1, 102.8
(5C, C1b, C1c, C1d, C1e, C1f); 84.5, 84.3 (5C, C3a, C3b, C3c, C3d,
C3e); 76.3, 75.9 (6C, C3f, C5a, C5b, C5c, C5d, C5e, C5f); 73.8,
73.6 (6C, C2a, C2b, C2c, C2d, C2e, C2f); 69.9 (C4f); 68.4 (4C, C4b,
C4c, C4d, C4e); 61.0 (5C, C6b, C6c, C6d, C6e, C6f).
TABLE-US-00008 HRMS (ESI.sup.+): [M + Na].sup.+
C.sub.36H.sub.62NaO.sub.30 theoretical m/z: 997.3224 measured m/z:
997.3201
7.9/ Preparation of
.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-.beta.-D-glucopyranosyl-(1.fwdarw.3)-.beta.-D-glucopyranosyl-(1.fwdarw.3)-
-.beta.-D-glucopyranosyl-(1.fwdarw.3)-4-deoxy-D-mannitol (Product
Db)
[0360] Compound Cb (40 mg, 0.041 mmol) is dissolved in a
methanol/water mixture (4:1, v/v) then 10 equivalents of sodium
borohydride (15.5 mg, 0.411 mmol) are added. After stirring for a
week at ambient temperature, the medium is neutralized by the
addition of a few drops of acetic acid, concentrated under reduced
pressure, coevaporated 3 times with 10 mL of a methanol/acetic acid
mixture (9:1, v/v) and finally with 3 times 10 mL of methanol.
Compound Db is obtained after purification on a gel permeation
column (Sephadex G-10, eluent: water) and freeze-drying of the
fractions collected.
[0361] Product Db: White Foam.
[0362] NMR .sup.1H (D.sub.2O, 400 MHz) .delta. (ppm):
Characteristic signals 3.97-3.90 (m, 1H, H3a); 1.70-1.40 (m, 2H,
H4a).
[0363] NMR .sup.13C (D.sub.2O, 100 MHz) .delta. (ppm):
Characteristic signals 76.3 (C3a); 71.0 (C2a, C5a); 66.1 (C6a);
63.0 (C1a); 25.3 (C4a).
[0364] Other Signals:
[0365] NMR .sup.1H (D.sub.2O, 400 MHz) .delta. (ppm): 4.70 (d, 3H,
H1, J.sub.H1-H2=8.0 Hz); 4.66 (d, 2H, H1, J.sub.H1-H2=8.0 Hz);
3.87-3.76 (m, 9H, H1a, H2a, H5a, H6a, H6b, H6c, H6d, H6e, H6f);
3.72-3.51 (m, 12H, H1'a, H3a, H3b, H3c, H3d, H3e, H6'a, H6'b, H6'c,
H6'd, H6'e, H6'f); 3.50-3.36 (m, 14H, H2b, H2c, H2d, H2e, H3f, H4b,
H4c, H4d, H4e, H5b, H5c, H5d, H5e, H5f); 3.31 (t, 1H, H4f,
J.sub.H3f-H4f=J.sub.H4f-H5f=9.3 Hz); 3.26 (dd, 1H, H2f,
J.sub.H1f-H2f=8.0 Hz, J.sub.H2f-H3f=9.3 Hz).
[0366] NMR .sup.13C (D.sub.2O, 100 MHz) .delta. (ppm): 103.1, 102.8
(5C, C1b, C1c, C1d, C1e, C1f); 84.5, 84.3 (4C, C3b, C3c, C3d, C3e);
76.3 (C5f); 75.9 (5C, C3f, C5a, C5b, C5c, C5d, C5e); 73.8, 73.6
(5C, C2b, C2c, C2d, C2e, C2f); 69.9 (C4f); 68.4, 68.2 (4C, C4b,
C4c, C4d, C4e); 61.0 (5C, C6b, C6c, C6d, C6e, C6f).
TABLE-US-00009 HRMS (ESI.sup.+): [M + Na].sup.+
C.sub.36H.sub.64NaO.sub.30 theoretical m/z: 999.3380 measured m/z:
999.3411
[0367] Examples 8 to 11 show the biological activities of compounds
of the invention.
[0368] In these examples, the tested compounds are:
LAM5: laminaripentaose, LAM7: laminariheptaose, A2 is the compound
prepared according to Example 3, A0 represents the following
compound:
##STR00029##
The control used is PBS. The dosages used are 100 .mu.g/mouse.
Example 8
Effect of LAM5, LAM7, A2 and A0 on the Phagocytosis of Peripheral
Blood Cells
[0369] One or other of a control, LAM5, LAM7, A0 or A2O were
injected, by peritoneal route, into a group of Balb/c mice (Jackson
laboratory, Bar Harbor, Me., USA). 24 hours after the injection the
mice were sacrificed. The peripheral blood was recovered from the
orbital plexus in heparin (5 IU/ml) (Sigma).
[0370] After counting, a test for phagocytosis of HEMA particles
(synthetic microspheres prepared from 2-hydroxyethylmethacrylate
copolymer) was carried out as described in: Rembaum et al., 1976,
Vetvicka et al. 1982, Bilej et al., 1989. 0.1 ml of heparinized
fresh blood was added to 0.05 ml of diluted HEMA particles
(5.times.108/ml) and incubated for 60 minutes at 37.degree. C.
under occasional moderate stirring.
[0371] At the end of the incubation, the cell suspension was
smeared onto microscope slides. The smears were evaluated under an
optical microscope after Giemsa staining.
[0372] Cells surrounding at least three particles were considered
to be positive.
[0373] The average results are represented graphically in FIG. 1.
They clearly show that both A0 and A2 strongly stimulate
phagocytosis in the monocytes and the granulocytes.
Example 9
Effect of the Oligosaccharides on the Phagocytosis of Cells
Obtained from the Peritoneal Cavity
[0374] One or other of a control, LAM7, LAM5, A0 or A2 were
injected, by intraperitoneal route, into a group of Balb/c mice
(Jackson laboratory, Bar Harbor, Me., USA).
[0375] After 24 hours, the mice were sacrificed, the peritoneal
cells were collected in a Hanks medium (Sigma).
[0376] After counting of the cells in a haemocytometer, the
peritoneal cells were diluted to 1.times.10.sup.7 in an RPMI 1640
medium (Sigma) with 5% foetal calf serum (Hyclone, Logan, Utah,
USA). 2.times.10.sup.6 cells in 0.2 ml of RPMI 1640 medium with 5%
foetal calf serum added were mixed with the same volume of HEMA
particles (5.times.10.sup.8/ml).
[0377] The suspension was incubated for 60 minutes at 37.degree. C.
under occasional moderate stirring. The incubation was stopped by
centrifugation (150 g for 5 minutes) and the pellet was replaced in
suspension.
[0378] The macrophages with ingested particles were counted under
an optical microscope in smears stained with Accustain (modified
Wright stain, Sigma).
[0379] The cells surrounding at least six particles were considered
as being positive.
[0380] The average results are represented graphically in FIG. 2.
They show that both A0 and A2 strongly stimulate phagocytosis both
in the monocytes and the granulocytes of peritoneal
macrophages.
Example 10
Effect of A0 and A2 on a Differential Count in the Blood
[0381] By using the same experimental groups as in Example 8, two
additional microscope slides were prepared from each experimental
sample. After Giemsa staining, the presence of individual types of
cells i.e. monocytes, lymphocytes and granulocytes were evaluated
for each slide with an optical microscope.
[0382] The average results are represented graphically in FIG. 3.
They show that both A0 and A2 increase the number of monocytes and
granulocytes in the peripheral blood.
Example 11
Effect of A0 and A2 on a Differential Count in the Peritoneal
Cells
[0383] By using the same experimental groups as in Example 8, two
additional microscope slides of each experimental sample were
prepared. After Giemsa staining, the presence of individual types
of cells i.e. macrophages, lymphocytes and mastocytes were
evaluated for each slide with an optical microscope.
[0384] The average results are represented graphically in FIG. 4.
They show that both A0 and A2 increase the number of monocytes and
granulocytes in the peritoneal cavity.
TABLE-US-00010 TABLE 1a NMR .sup.1H chemical shifts and
multiplicity of the monosaccharides corresponding to the formula:
##STR00030## Compound .delta. (ppm)/J.sub.Hn-Hn+1 R.sup.2
H-1/J.sub.1.2 H-2/J.sub.2.3 H-3/J.sub.3.4 -4/J.sub.4.5 -5/J.sub.5.6
H-6/J.sub.6.6' 6'/J.sub.6'.5 NAP 4.51/8.1 5.12/9.1 3.76/9.1 83/9.1
.44/5.1 .39/10.4 85/10.4 H 4.56/8.0 4.99/9.1 3.61/9.1 86/9.1
.42/5.1 .36/10.4 80/10.4 NAP 4.50/7.6 3.70/8.9 3.72/8.9 76/9.9
.45/5.1 .38/10.2 84/10.2 indicates data missing or illegible when
filed
TABLE-US-00011 TABLEAU 1b NMR .sup.13C chemical shifts and
multiplicity of the monosaccharides corresponding to the formula:
##STR00031## Compound .delta. (ppm) n .degree. R.sup.1 R.sup.2 C-1
C-2 C-3 C-4 C-5 C-6 3 Lev NAP 00.1 73.2 78.1 81.5 66.2 68.6 4 Lev H
99.8 74.5 72.0 80.5 66.2 68.5 10 H NAP 02.2 74.5 80.0 81.3 66.5
68.7
TABLE-US-00012 TABLEAU 2a NMR .sup.1H chemical shifts and
multiplicity of the monosaccharides corresponding to the formula:
##STR00032## .delta. (ppm)/multiplicity Compound Unit (b) n
.degree. R.sup.1 R.sup.2 /J.sub.1.2 /J.sub.2.3 /J.sub.3.4
/J.sub.4.5 /J.sub.5.6 /J.sub.6.6' /J.sub.6',5 5 ev AP /7.1 /7.1
/9.1 /9.1 3/4.9 /10.4 /10.4 6 ev H /6.8 /7.1 /9.3 /9.3 6/5.1 /10.6
/10.6 1 H AP /7.1 /7.5 /9.1 /9.1 8/4.9 /10.4 /10.4 .delta.
(ppm)/multiplicity Compound Unit (a) n .degree. R.sup.1 R.sup.2
/J.sub.1.2 /J.sub.2.3 /J.sub.3.4 /J.sub.4.5 /J.sub.5.6 /J.sub.6.6'
/J.sub.6',5 5 ev AP /7.8 /8.9 /8.9 /8.9 3/4.9 /10.4 /10.4 6 ev H
/7.5 /8.4 /9.1 /9.1 9/4.8 /10.6 /10.6 1 H AP /7.8 /8.6 /9.0 /9.0
5/5.1 /10.6 /10.6 indicates data missing or illegible when
filed
TABLE-US-00013 TABLEAU 2b NMR .sup.13C chemical shifts and
multiplicity of the monosaccharides corresponding to the formula:
##STR00033## .delta. (ppm) Compound Unit (b) n .degree. R.sup.1
R.sup.2 C-1 C-2 C-3 C-4 C-5 C-6 5 ev AP 0.1 4.0 8.1 0.7 5.9 8.6 6
ev H 0.0 5.6 2.5 0.5 5.8 8.6 1 H AP 1.1 4.0 7.8 1.3 6.0 8.6 .delta.
(ppm) Compound Unit (a) n .degree. R.sup.1 R.sup.2 C-1 C-2 C-3 C-4
C-5 C-6 5 ev AP 9.8 2.9 7.5 9.7 6.3 8.6 6 ev H 9.7 2.9 7.6 9.6 6.2
8.6 1 H AP 2.1 4.1 9.2 1.1 6.5 8.7 indicates data missing or
illegible when filed
TABLE-US-00014 TABLEAU 3a NMR .sup.1H chemical shifts and
multiplicity of the monosaccharides corresponding to the formula:
##STR00034## .delta. (ppm)/multiplicity Compound Unit (c) .degree.
R.sup.1 R.sup.2 /J.sub.1.2 /J.sub.2.3 /J.sub.3.4 /J.sub.4.5
/J.sub.5.6 /J.sub.6.6' /J.sub.6',5 7 ev AP 14/ 39/ 05/ -3.87/
-3.52/ 27/ 76/ .5 .2 .0 d .1 .6 .6 8 ev 14/ 20/ 94/ 62/ 49/ 19/ 66/
.8 .8 .8 .8 .9 .4 .4 2 AP 12/ 40/ 92/ 95/ 53/ 29/ 79/ .0 .0 .0 .0
.9 .4 .4 .delta. (ppm)/multiplicity Compound Unit (b) .degree.
R.sup.1 R.sup.2 /J.sub.1.2 /J.sub.2.3 /J.sub.3.4 /J.sub.4.5
/J.sub.5.6 /J.sub.6.6' /J.sub.6',5 7 ev AP 12/ -5.11/ -4.08/ -3.87/
-3.60/ -4.08/ 49/ .2 d d d d .4 .4 8 ev 11/ -5.10/ -4.03/ -4.03/
-3.57/ -4.03 44/ .0 d d d d .0 .0 2 AP 13/ 18/ 02/ 21/ 64/ 09/ 52/
.2 .2 .0 .2 .6 .0 .0 .delta. (ppm)/multiplicity Compound Unit (a)
.degree. R.sup.1 R.sup.2 /J.sub.1.2 /J.sub.2.3 /J.sub.3.4
/J.sub.4.5 /J.sub.5.6 /J.sub.6.6' /J.sub.6',5 7 ev AP 33/ 60/ 92/
02/ 31/ 27/ 58/ .0 .8 .3 .3 .1 .4 .4/ 8 ev 29/ 64/ 89/ 98/ 27/ 23/
53/ .8 .2 .2 .2 .9 .4 .4 2 AP 27/ 85/ 73/ -3.32/ -3.32/ 32/ 75/ .0
.0 .8 d .8 .0 .0 indicates data missing or illegible when filed
TABLE-US-00015 TABLEAU 3b NMR .sup.13C chemical shifts and
multiplicity of the monosaccharides corresponding to the formula:
##STR00035## .delta. (ppm) Compound Unit (c) Unit (b) .degree.
R.sup.1 R.sup.2 -1 -2 -3 -4 -5 -6 -1 -2 -3 -4 -5 -6 7 ev AP .0 .2
.1 .4 .1 .7 .3 .2 .6 .1 .1 .7 ev .8 .6 .4 .8 .0 .6 .4 .3 .2 .7 .1
.7 2 AP .6 .3 .2 .3 .1 .6 .6 .5 .9 .6 .1 .7 .delta. (ppm) Compound
Unit (a) .degree. R.sup.1 R.sup.2 -1 -2 -3 -4 -5 -6 7 ev AP .6 .8
.5 .7 .2 .5 ev .6 .7 .7 .6 .2 .5 2 AP 2.0 .7 .7 .8 .6 .6 indicates
data missing or illegible when filed
TABLE-US-00016 TABLEAU 3'a NMR .sup.1H chemical shifts and
multiplicity of the monosaccharides corresponding to the formula:
##STR00036## .delta. (ppm)/multiplicity Compound Unit (c) n
.degree. R.sup.1 /J.sub.1.2 /J.sub.2.3 /J.sub.3.4 /J.sub.4.5
/J.sub.5.6 /J.sub.6.6' /J.sub.6',5 18 Bz 97/ 34/ 11/ 94/ 48/ 24/
74/ .5 .5 .3 .3 .8 0.4 0.4 19 H 57/ -3.68/ 83/ 66/ 38/ 30 75/ .7 .5
.5 .5 .8 0.4 0.4 .delta. (ppm)/multiplicity Compound Unit (b) n
.degree. R.sup.1 /J.sub.1.2 /J.sub.2.3 /J.sub.3.4 /J.sub.4.5
/J.sub.5.6 /J.sub.6.6' /J.sub.6',5 18 Bz 84/ 26/ 82/ 05/ 55/ 18/
74/ .2 .2 .5 d .6 0.2 .02 19 H 57/ -3.68/ .68/m .68/m 38/ 30/ 76/
.1 d .8 0.4 0.4 .delta. (ppm)/multiplicity Compound Unit (a) n
.degree. R.sup.1 /J.sub.1.2 /J.sub.2.3 /J.sub.3.4 /J.sub.4.5
/J.sub.5.6 /J.sub.6.6' /J.sub.6',5 18 Bz 35/ 82/ 70/ 94/ 25/ 32/
76/ .7 0.2 .3 .3 .8 0.4 .4t 92 H 59/ 19/ 92/ 12/ /5.1 37/ 92/ 1.0
.7 .5 .5 0.6 0.6 indicates data missing or illegible when filed
TABLE-US-00017 TABLEAU 3'b NMR .sup.13C chemical shifts and
multiplicity of the monosaccharides corresponding to the formula:
##STR00037## .delta. (ppm) ompound Unit (c) Unit (b) .degree.
R.sup.1 -1 -2 -3 -4 -5 -6 -1 -2 -3 -4 -5 -6 z 0.1 .5 .8 .9 .0 .6 .8
.0 .0 .3 .1 .6 5.7 .0 .6 .6 .9 .3 1.3 .1 .9 .8 .7 .5 .delta. (ppm)
ompound Unit (a) .degree. R.sup.1 -1 -2 -3 -4 -5 -6 z .3 .9 .0 .5
.0 .4 .1 .4 .9 .3 .1 .4 indicates data missing or illegible when
filed
* * * * *